Delta15 desaturases suitable for altering levels of polyunsaturated fatty acids in oilseed plants and oleaginous yeast

ABSTRACT

The present invention relates to fungal Δ-15 fatty acid desaturases that are able to catalyze the conversion of linoleic acid (18:2, LA) to alpha-linolenic acid (18:3, ALA). Nucleic acid sequences encoding the desaturases, nucleic acid sequences which hybridize thereto, DNA constructs comprising the desaturase genes, and recombinant host plants and microorganisms expressing increased levels of the desaturases are described. Methods of increasing production of specific omega-3 and omega-6 fatty acids by over-expression of the Δ-15 fatty acid desaturases are also described herein.

This application claims the benefit of U.S. Provisional Application No.60/519191, filed Nov. 12, 2003.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to the identification of nucleic acid fragmentsencoding Δ-15 fatty acid desaturase enzymes useful for disrupting orenhancing the production of polyunsaturated fatty acids in plants andorganisms, including those microorganisms known as as oleaginous yeast.

BACKGROUND OF THE INVENTION

It has long been recognized that certain polyunsaturated fatty acids, orPUFAs, are important biological components of healthy cells. Forexample, such PUFAs are recognized as:

-   -   “Essential” fatty acids that can not be synthesized de novo in        mammals and instead must be obtained either in the diet or        derived by further desaturation and elongation of linoleic acid        (LA) or -α-linolenic acid (ALA);    -   Constituents of plasma membranes of cells, where they may be        found in such forms as phospholipids or triglycerides;    -   Necessary for proper development, particularly in the developing        infant brain, and for tissue formation and repair; and,    -   Precursors to several biologically active eicosanoids of        importance in mammals, including prostacyclins, eicosanoids,        leukotrienes and prostaglandins.

In the 1970's, observations of Greenland Eskimos linked a low incidenceof heart disease and a high intake of long-chain omega-3 PUFAs(Dyerberg, J. et al., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg,J. et al., Lancet 2(8081):117-119 (Jul. 15, 1978)). More recent studieshave confirmed the cardiovascular protective effects of omega-3 PUFAs(Shimokawa, H., World Rev Nutr Diet, 88:100-108 (2001); von Schacky, C.,and Dyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). Further, it hasbeen discovered that several disorders respond to treatment withomega-3fatty acids, such as the rate of restenosis after angioplasty,symptoms of inflammation and rheumatoid arthritis, asthma, psoriasis andeczema. Gamma-linolenic acid (GLA, an omega-6 PUFA) has been shown toreduce increases in blood pressure associated with stress and to improveperformance on arithmetic tests. GLA and dihomo-gamma-linolenic acid(DGLA, another omega-6 PUFA) have been shown to inhibit plateletaggregation, cause vasodilation, lower cholesterol levels and inhibitproliferation of vessel wall smooth muscle and fibrous tissue (Brenneret al., Adv. Exp. Med. Biol. 83: 85-101 (1976)). Administration of GLAor DGLA, alone or in combination with eicosapentaenoic acid (EPA, anomega-3 PUFA), has been shown to reduce or prevent gastrointestinalbleeding and other side effects caused by non-steroidalanti-inflammatory drugs (U.S. Pat. No. 4,666,701). Further, GLA and DGLAhave been shown to prevent or treat endometriosis and premenstrualsyndrome (U.S. Pat. No. 4,758,592) and to treat myalgicencephalomyelitis and chronic fatigue after viral infections (U.S. Pat.No. 5,116,871). Other evidence indicates that PUFAs may be involved inthe regulation of calcium metabolism, suggesting that they may be usefulin the treatment or prevention of osteoporosis and kidney or urinarytract stones. Finally, PUFAs can be used in the treatment of cancer anddiabetes (U.S. Pat. No. 4,826,877; Horrobin et al., Am. J. Clin. Nutr.57 (Suppl.): 732S-737S (1993)).

PUFAs are generally divided into two major classes (consisting of theomega-6 and the omega-3 fatty acids) that are derived by desaturationand elongation of the essential fatty acids, LA and ALA, respectively.Despite a variety of commercial sources of PUFAs from natural sources[e.g., seeds of evening primrose, borage and black currants; filamentousfungi (Mortierella), Porphyridium (red alga), fish oils and marineplankton (Cyclotella, Nitzschia, Crypthecodinium)], there are severaldisadvantages associated with these methods of production. First,natural sources such as fish and plants tend to have highlyheterogeneous oil compositions. The oils obtained from these sourcestherefore can require extensive purification to separate or enrich oneor more of the desired PUFAs. Natural sources are also subject touncontrollable fluctuations in availability (e.g., due to weather,disease, or over-fishing in the case of fish stocks); and, crops thatproduce PUFAs often are not competitive economically with hybrid cropsdeveloped for food production. Large-scale fermentation of someorganisms that naturally produce PUFAs (e.g., Porphyridium, Mortierella)can also be expensive and/or difficult to cultivate on a commercialscale.

As a result of the limitations described above, extensive work has beenconducted toward: 1.) the development of recombinant sources of PUFAsthat are easy to produce commercially; and 2.) modification of fattyacid biosynthetic pathways, to enable production of desired PUFAs. Forexample, advances in the isolation, cloning and manipulation of fattyacid desaturase and elongase genes from various organisms have been madeover the last several years. Knowledge of these gene sequences offersthe prospect of producing a desired fatty acid and/or fatty acidcomposition in novel host organisms that do not naturally produce PUFAs.The literature reports a number of examples in Saccharomyces cerevisiae,such as: Domergue, F., et al. (Eur. J. Biochem. 269:41054113 (2002)),wherein two desaturases from the marine diatom Phaeodactylum tricomutumwere cloned into S. cerevisiae, leading to the production of EPA;Beaudoin F., et al. (Proc. Natl. Acad. Sci. U.S.A. 97(12):6421-6(2000)), wherein the omega-3 and omega-6 PUFA biosynthetic pathways werereconstituted in S. cerevisiae, using genes from Caenorhabditis elegans;Dyer, J. M., et al. (Appl. Eniv. Microbiol., 59:224-230 (2002)), whereinplant fatty acid desaturases (FAD2 and FAD3) were expressed in S.cerevisiae, leading to the production of ALA; and, U.S. Pat. No.6,136,574 (Knutzon et al., Abbott Laboratories), wherein one desaturasefrom Brassica napus and two desaturases from the fungus Mortierellaalpina were cloned into S. cerevisiae, leading to the production of LA,GLA, ALA and STA.

There remains a need, however, for an appropriate plant and/or microbialsystem in which these types of genes can be expressed to provide foreconomical production of commercial quantities of one or more PUFAs.Additionally, a need exists for oils enriched in specific PUFAs, notablyEPA and DHA.

One class of microorganisms that has not been previously examined as aproduction platform for PUFAs are the oleaginous yeast. These organismscan accumulate oil up to 80% of their dry cell weight. The technologyfor growing oleaginous yeast with high oil content is well developed(for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol.16:119-206 (1982)), and may offer a cost advantage compared tocommercial micro-algae fermentation for production of ω-3- or ω-6 PUFAs.Whole yeast cells may also represent a convenient way of encapsulatingomega-3- or omega-6 PUFA-enriched oils for use in functional foods andanimal feed supplements.

Despite the advantages noted above, most oleaginous yeast are naturallydeficient in omega-6 PUFAs, since naturally produced PUFAs in theseorganisms are usually limited to 18:2 fatty acids. Thus, the problem tobe solved is to develop an oleaginous yeast that accumulates oilsenriched in omega-3 and/or omega-6 fatty acids. Toward this end, it isnot only necessary to introduce the required desaturases and elongasesthat allow for the synthesis and accumulation of omega-3 and/or omega-6fatty acids in oleaginous yeast, but also to increase the availabilityof the 18:3 substrate (i.e., ALA for ω-3 production). Generally, theavailability of this substrate is controlled by the activity of Δ-15desaturases that catalyze the conversion of LA to ALA.

There were a variety of known Δ-15 desaturases disclosed in the publicliterature, including those from photosynthetic organisms (e.g., plants)and Caenorhabditis elegans at the time that the instant invention wasmade. These desaturases are not known to be effective for altering fattyacid composition in oleaginous yeast and are not preferred for use inoleaginous yeast. Furthermore, heterologous expression of thesedesaturases in the non-oleaginous yeast Saccharomyces cerevisiae hasresulted in production of less than 5% ALA (Reed, D. et al. PlantPhysiol. 122:715-720 (2000); Meesapyodsuk, D. et al. Biochem.39:11948-11954 (2000); WO 2003/099216). Thus, there is need for theidentification and isolation of genes encoding Δ-15 desaturases that areable to support production of high levels of 18:3 (ALA) and higherratios of omega-3 to omega-6 fatty acids in oleaginous microorganisms(e.g., oleaginous yeast) for use in the production of PUFAs.

The instant invention concerns, inter alia, isolation of the geneencoding a Δ-15 desaturase from the fungus Fusarium moniliforme anddemonstrating surprisingly efficient conversion of 18:2 (LA) to 18:3(ALA) upon expression in an oleaginous yeast. Orthologs of this Δ-15desaturase were identified in Magnaporthe grisea, Fusarium graminearium,Aspergillus nidulans and Neurospora crassa. Upon further experimentalanalysis of the Fusarium moniliforme and Magnaporthe grisea desaturases'activity, however, it was surprisingly shown that both A-15 desaturasesalso have Δ-12 desaturase activity (and thus the enzymes arecharacterized herein as having bifunctional Δ-12/Δ-15 desaturaseactivity).

In addition to the interest in oleaginous yeast as a production platformfor PUFAs, there has also been interest in plants as an alternativeproduction platform for PUFAs.

WO 02/26946, published Apr. 4, 2002, describes isolated nucleic acidfragments encoding FAD4, FAD5, FAD5-2 and FAD6 fatty acid desaturasefamily members which are expressed in LCPUFA-producing organisms, e.g.,Thraustochytrium, Pythium irregulare, Schizichytrium andCrypthecodinium. It is indicated that constructs containing thedesaturase genes can be used in any expression system including plants,animals, and microorganisms for the production of cells capable ofproducing LCPUFAs.

WO 02/26946, published Apr. 4, 2002, describes FAD4, FAD5, FAD5-2, andFAD6 fatty acid desaturase members and uses thereof to produce longchain polyunsaturated fatty acids.

WO 98/55625, published Dec. 19, 1998, describes the production ofpolyunsaturated fatty acids by expression of polyketide-like synthesisgenes in plants.

WO 98/46764, published Oct. 22, 1998, describes compositions and methodsfor preparing long chain fatty acids in plants, plant parts and plantcells which utilize nucleic acid sequences and constructs encoding fattyacid desaturases, including Δ-5 desaturases, Δ-6 desaturases and Δ-12desaturases.

U.S. Pat. No. 6,075,183, issued to Knutzon et al. on Jun. 13, 2000,describes methods and compositions for synthesis of long chainpolyunsaturated fatty acids in plants.

U.S. Pat. No. 6,459,018, issued to Knutzon on Oct. 1, 2002, describes amethod for producing stearidonic acid in plant seed utilizing aconstruct comprising a DNA sequence encoding a Δ-six desaturase.

Spychalla et al., Proc. Natl. Acad. Sci. USA, Vol.94,1142-1147 (February1997), describes the isolation and characterization of a cDNA from C.elegans that, when expressed in Arabidopsis, encodes a fatty aciddesaturase which can catalyze the introduction of an omega-3 double bondinto a range of 18- and 20-carbon fatty acids.

WO 2004/071467 published on Aug. 26, 2004 describes the production ofvery long chain polyunsaturated fatty acids in plants.

SUMMARY OF THE INVENTION

In one embodiment, the invention concerns a recombinant construct foraltering the total fatty acid profile of mature seeds of an oilseedplant to produce an oil having an omega 3 to omega 6 ratio greater than0.4, said construct comprising an isolated nucleic acid fragmentselected from the group consisting of:

-   -   (a) an isolated nucleic acid fragment encoding all or part of        the amino acid sequence as set forth in SEQ ID NO:2;    -   (b) an isolated nucleic acid fragment that hybridizes with (a)        when washed with : 0.1×SSC, 0.1% SDS, 65° C.;    -   (c) an isolated nucleic acid fragment encoding an amino acid        sequence having at least 46.2% sequence identity with the amino        acid sequences set forth in SEQ ID NOs:2, 6, 10, 14,18 based on        the Clustal V method of alignment; or    -   (d) an isolated nucleic acid fragment that is completely        complementary to (a), (b), or (c)        wherein said isolated nucleic acid fragment is operably linked        to at least one regulatory sequence.

In a second embodiment, this invention concerns oilseed plants, plantcells, plant tissues or plant parts comprising in their genomes therecombinant construct of the invention.

In a third embodiment, this inventions also concerns seeds obtained fromsuch plants, oil obtained from these seeds and by-products obtained fromthe processing of this oil.

In a fourth embodiment, this invention concerns use of the oil of theinvention in food, animal feed or an industrial application and use ofthe by-products of the invention in food or animal feed.

In a fifth embodiment, this invention concerns a method for increasingthe ratio of omega-3 fatty acids to omega-6 fatty acids in an oilseedplant comprising:

-   -   a) transforming an oilseed plant cell of with the recombinant        construct of the invention;    -   b) regenerating an oilseed plant from the transformed plant cell        of step (a);    -   c) selecting those transformed plants having an increased ratio        of omega-3 fatty acids to omega-6 fatty acid compared to the        ratio of omega-3 fatty acids to omega-6 fatty acid in an        untransformed plant.

In a sixth embodiment, this invention concerns oilseed plants made bythis method, seeds obtained from such plants, oil obtained from theseseeds, use of this oil in food or animal feed, by-products obtained fromthe processing of this oil and use of these by-products in food oranimal feed.

In a seventh embodiment, this invention concerns a method for producingalpha-linolenic acid in seed of an oilseed plant wherein thealpha-linolenic acid content of the oil in the seed is at least 25% ofthe total fatty acid content of the seed oil, said method comprising:

-   -   a) transforming an oilseed plant cell of with the recombinant        construct of the invention;    -   b) regenerating an oilseed plant from the transformed plant cell        of step (a);    -   c) selecting those transformed plants having at least 25%        alpha-linolenic acid of the total fatty acid content of the seed        oil.

In an eighth embodiment, this invention concerns oilseed plants made bythis method, seeds obtained from such plants, oil obtained from theseseeds, use of this oil in food or animal feed, by-products obtained fromthe processing of this oil and use of these by-products in food oranimal feed.

Alternatively, the invention provides an isolated nucleic acid fragmentencoding a fungal Δ15 desaturase enzyme, selected from the groupconsisting of:

-   -   (a) an isolated nucleic acid fragment encoding the amino acid        sequence as set forth in SEQ ID NO:2;    -   (b) an isolated nucleic acid fragment that hybridizes with (a)        under the following hybridization conditions: 0.1×SSC, 0.1% SDS,        65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%        SDS; or,        an isolated nucleic acid fragment that is complementary to (a)        or (b).

Alternatively the invention provides an isolated nucleic acid fragmentcomprising a first nucleotide sequence encoding a Δ15 desaturase enzymeof at least 402 amino acids that has at least 86% identity based on theClustal method of alignment when compared to a polypeptide having thesequence as set forth in SEQ ID NO:2; or a second nucleotide sequencecomprising the complement of the first nucleotide sequence.

Additionally the invention provides polypeptides encoded by the nucleicacids described herein as well as geneic chimera and transformed hostcomprising the same. Preferred host cells for use in the inventioninclude, but are not limited to plants, algae, bacteria, yeast and fungi

In another embodiment the invention provides a method for the productionof α-linolenic acid comprising:

-   -   a) providing a host cell comprising:        -   (i) an isolated nucleic acid fragment encoding a protein            having Δ15 desaturase activity that has at least 46.2%            identity based on the Clustal method of alignment when            compared to a polypeptide having the sequence as set forth            in SEQ ID NO:2; and        -   (ii) a source of linoleic acid;    -   b) growing the host cell of step (a) under conditions wherein        the nucleic acid fragment encoding a protein having Δ15        desaturase activity is expressed and the linoleic acid is        converted to α-linolenic acid; and    -   c) optionally recovering the α-linolenic acid of step (b).

Similarly the invention provides a method for the production ofα-linolenic acid comprising:

-   -   a) providing a host cell comprising:        -   (i) an isolated nucleic acid fragment encoding a protein            having Δ15 desaturase activity that has at least 46.2%            identity based on the Clustal method of alignment when            compared to a polypeptide having the sequence as set forth            in SEQ ID NO:2; and        -   (ii) a source of oleic acid;    -   b) growing the host cell of step (a) under conditions wherein        the nucleic acid fragment encoding a protein having Δ15        desaturase activity is expressed and the oleic acid is converted        to α-linolenic acid; and    -   c) optionally recovering the α-linolenic acid of step (b).

Alternatively the invention provides a method for the production of ω-3fatty acids in a host cell comprising:

-   -   a) providing a host cell comprising:        -   (i) an isolated nucleic acid fragment encoding a protein            having Δ15 desaturase activity that has at least 46.2%            identity based on the Clustal method of alignment when            compared to a polypeptide having the sequence as set forth            in SEQ ID NO:2; and        -   (ii) genes encoding a functional ω-3/ ω-6 fatty acid            biosynthetic pathway;    -   b) providing a source of desaturase substrate consisting of        oleic acid;    -   c) growing the host cell of step (a) with the desaturase        substrate of step (b) under conditions wherein ω-3 fatty acids        are produced; and    -   d) optionally recovering the ω-3 fatty acids of step (c).

In an alternate embodiment the invention provides a method of increasingthe ratio of ω-3 fatty acids to ω-6 fatty acids in a host cell producingω-3 fatty acids and ω-6 fatty acids comprising:

-   -   a) providing a host cell producing ω-3 fatty acids and ω-6 fatty        acids;    -   b) introducing into the host cell of (a) an isolated nucleic        acid fragment encoding a protein having at least 46.2% identity        based on the Clustal method of alignment when compared to a        polypeptide having the sequence as set forth in SEQ ID NO:2,        wherein the polypeptide binds both oleic acid and linolenic acid        as an enzyme substrate, wherein the ratio of ω-3 fatty acids to        ω-6 fatty acids are increased.

Additionally the invention provides microbial oils produced by themethods of the invention.

In yet another embodiment, the invention concerns a recombinantconstruct for altering the total fatty acid profile of mature seeds ofan oilseed plant to produce an oil having an omega 3 to omega 6 ratiogreater than 2, wherein said oil has an eicosapentaenoic acid contentgreater than 2%, said construct comprising an isolated nucleic acidfragment selected from the group consisting of:

-   -   (a) an isolated nucleic acid fragment encoding all or part of        the amino acid sequence as set forth in SEQ ID NO:2;    -   (b) an isolated nucleic acid fragment that hybridizes with (a)        when washed with 0.1×SSC, 0.1% SDS, 65° C.;    -   (c) an isolated nucleic acid fragment encoding an amino acid        sequence having at least 46.2% sequence identity with the amino        acid sequences set forth in SEQ ID NOs:2, 6, 10, 14, 18 based on        the Clustal V method of alignment; or    -   (d) an isolated nucleic acid fragment that is completely        complementary to (a), (b), or (c)        wherein said isolated nucleic acid fragment is operably linked        to at least one regulatory sequence.

In a further embodiment, this invention concerns oilseed plants, plantcells, plant tissues, or plant parts comprising in their genomes therecombinant construct of the invention. The invention also concerns theseeds obtained from such plants, oil obtained from these seeds, use ofthis oil in food or animal feed, by-products obtained from theprocessing of this oil and use of these by-products in food or animalfeed.

Additionally the invention provides microbial oils produced by themethods of the invention.

In another embodiment, the present invention concerns a method forproducing eicosapentaenoic acid in seed of an oilseed plant to producean oil having an omega 3 to omega 6 ratio greater than 2, wherein saidoil has an eicosapentaenoic acid content greater than 2% of the totalfatty acid content of the seed oil, said method comprising:

-   -   a) transforming an oilseed plant cell of with the recombinant        construct of the present invention;    -   b) regenerating an oilseed plant from the transformed plant cell        of step (a);    -   c) selecting those transformed plants having at least 2%        eicosapentaenoic acid of the total fatty acid content of the        seed oil.

In a further embodiment, this invention concerns oilseed plants, plantcells, plant tissues, or plant parts comprising in their genomes therecombinant construct of the invention. The invention also concerns theseeds obtained from such plants, oil obtained from these seeds, use ofthis oil in food or animal feed, by-products obtained from theprocessing of this oil and use of these by-products in food or animalfeed.

Additionally the invention provides microbial oils produced by themethods of the invention.

Biological Deposits

The following plasmids have been deposited with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209, and bears the following designation, accession number anddate of deposit. Plasmid Accession Number Date of Deposit pKR274 ATCCPTA-4988 Jan. 30, 2003 pKKE2 ATCC PTA-4987 Jan. 30, 2003 pKR578 ATCCPTA-XXXX Nov. 4, 2004 pKR585 ATCC PTA-XXXX Nov. 4, 2004

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 shows a schematic illustration of the biochemical mechanism forlipid accumulation in oleaginous yeast.

FIG. 2 illustrates the omega-3 and omega-6 fatty acid biosyntheticpathways.

FIG. 3 illustrates the construction of the plasmid vector pY5 for geneexpression in Yarrowia lipolytica.

FIG. 4 shows a phylogenetic tree of proteins from different filamentousfungi (i.e., Aspergillus nidulans, Fusarium moniliforme, F.graminearium, Magnaporthe grisea and Neurospora crassa) having homologyto the Yarrowia lipolytica Δ12 desaturase enzyme, and created usingMegalign DNASTAR software.

FIG. 5 shows a pairwise comparison (% Identity) between and amongproteins from different filamentous fungi having homology to theYarrowia lipolytica Δ12 desaturase enzyme using a ClustalW analysis(Megalign program of DNASTAR sofware).

FIG. 6 is a schematic depiction of plasmid pKR578 (see Example 11).

FIG. 7 is a schematic depiction of plasmid pKR585 (see Example 13).

FIG. 8 is a schematic depiction of plasmid pKR274 (see Example 14).

FIG. 9 is a schematic depiction of plasmid pKKE2 (see Example 15).

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1-20, 54 and 55 are ORFs encoding genes or proteins asidentified in Table 1. TABLE 1 Summary Of Desaturase Gene And ProteinSEQ ID Numbers ORF Nucleic acid Protein Description SEQ ID NO. SEQ IDNO. Fusarium moniliforme sub-family 1  1  2 desaturase (Δ15/Δ12desaturase) (1209 bp) (402 AA) Fusarium moniliforme sub-family 2  3  4desaturase (1434 bp) (477 AA) Aspergillus nidulans sub-family 1  5  6desaturase (Δ15 desaturase) (1206 bp) (401 AA) Aspergillus nidulanssub-family 2  7  8 desaturase (1416 bp) (471 AA) Magnaporthe griseasub-family 1  9 10 desaturase (Δ15 desaturase) (1185 bp) (394 AA)Magnaporthe grisea sub-family 2 11 12 desaturase (1656 bp) (551 AA)Neurospora crassa sub-family 1 13 14 desaturase (Δ15 desaturase) (1290bp) (429 AA) Neurospora crassa sub-family 2 15 16 desaturase (1446 bp)(481 AA) Fusarium graminearium sub-family 1 17 18 desaturase (Δ15desaturase) (1212 bp) (403 AA) Fusarium graminearium sub-family 2 19 20desaturase (1371 bp) (456 AA) Yarrowia lipolytica 54 55 Δ12 desaturase(1936 bp) (419 AA)

SEQ ID NOs:21 and 22 are primers TEF 5′ and TEF 3′, respectively, usedto isolate the TEF promoter.

SEQ ID NOs:23 and 24 are primers XPR 5′ and XPR 3′, respectively, usedto isolate the XPR2 transcriptional terminator.

SEQ ID NOs:25-36 correspond to primers YL5, YL6, YL9, YL10, YL7, YL8,YL3, YL4, YL1, YL2, YL61 and YL62, respectively, used for plasmidconstruction.

SEQ ID NO:37 corresponds to a 971 bp fragment designated as “GPDPro”,and identified as the putative glyceraldehyde-3-phosphate dehydrogenasepromoter in Yarrowia lipolytica.

SEQ ID NOs:38 and 39 are primers YL211 and YL212, respectively, used foramplifying a DNA fragment including theglyceraldehyde-3-phosphate-dehydrogenase (GPD) promoter of Yarrowialipolytica.

SEQ ID NOs:40 and 41 are primers GPDsense and GPDantisense,respectively, used for re-amplifying the GPD promoter.

SEQ ID NOs:42 and 44 are the degenerate primers identified as P73 andP76, respectively, used for the isolation of a Yarrowia lipolytica Δ12desaturase gene.

SEQ ID NOs:43 and 45 are the amino acid consensus sequences thatcorrespond to the degenerate primers P73 and P76, respectively.

SEQ ID NOs:46-49 correspond to primers P99, P100, P101 and P102,respectively, used for targeted disruption of the native Y. lipolyticaΔ12 desaturase gene.

SEQ ID NOs:50-53 correspond to primers P119, P120, P121 and P122,respectively, used to screen for targeted integration of the disruptedY. lipolytica Δ12 desaturase gene.

SEQ ID NOs:56 and 57 are primers P192 and P193, respectively, used toamplify the Fusarium moniliforme Δ15 desaturase (“Fm1”) coding region.

SEQ ID NO:58 corresponds to the codon-optimized translation initiationsite for genes optimally expressed in Yarrowia sp.

SEQ ID NOs:59-64 are primers P186, P187, P188, P189, P190 and P191,respectively, used to amplify the Magnaporthe grisea Δ15 desaturase(“Mg1”).

SEQ ID NOs:65-72 are primers PFg1UP1, PFg1LP1, PFg1UP2, PFg1LP2,PFg1UP3, PFg2LP3, PFg1UP4 and PFg1LP4, respectively, used to amplify theFusarium graminearium Δ15 desaturase (“Fg1”).

SEQ ID NO:73 is the multiple restriction enzyme site sequence introducedupstream of the Kti promoter as described in Example 6.

SEQ ID NO:74 sets forth the sequence of the soy albumin transcriptionterminator with restriction enzyme sites as described in Example 6.

SEQ ID NO:75 is the primer oSalb-12 used for amplification of thealbumin transcription terminator.

SEQ ID NO:76 is primer oSalb-13 used for amplification of the albumintranscription terminator.

SEQ ID NO:77 is the multiple restriction enzyme site sequence introducedin front of the beta-conglycinin promoter as described in Example 6.

SEQ ID NO:78 is the complete sequence of plasmid pKR578 described inExample 11 and FIG. 5.

SEQ. ID. NO:79 sets forth oligonucleotide primer GSP1 used to amplifythe soybean annexin promoter.

SEQ. ID. NO:80 sets forth oligonucleotide primer GSP2 used to amplifythe soybean annexin promoter.

SEQ. ID. NO:81 sets forth the sequence of the annexin promoter.

SEQ. ID. NO:82 sets forth oligonucleotide primer GSP3 used to amplifythe soybean BD30 promoter.

SEQ ID NO:83 sets forth oligonucleotide primer GSP4 used to amplify thesoybean BD30 promoter.

SEQ. ID. NO:84 sets forth the sequence of the soybean BD30 promoter.

SEQ. ID. NO:85 sets forth the sequence of the soybean β-conglycininβ-subunit promoter.

SEQ. ID. NO:86 sets forth oligonucleotide primer β-con oligo Bam used toamplify the promoter for soybean β-conglycinin β-subunit.

SEQ. ID. NO:87 sets forth oligonucleotide primer β-con oligo Not used toamplify the promoter for soybean β-conglycinin β-subunit.

SEQ. ID. NO:88 sets forth the sequence of the soybean glycinin Gly-1promoter.

SEQ. ID. NO:89 sets forth oligonucleotide primer glyoligo Bam used toamplify the Gly-1 promoter.

SEQ. ID. NO:90 sets forth oligonucleotide primer glyoligo Not used toamplify the Gly-1 promoter.

SEQ ID NO:91 is primer oKTi5 used for amplification of the Kti/NotI/Kti3′ cassette.

SEQ ID NO:92 is primer oKTi6 used for amplification of the Kti/NotI/Kti3′ cassette.

SEQ ID NO:93 is primer oSBD30-1 used for amplification of the soybeanBD30 3′ transcription terminator.

SEQ ID NO:94 is primer oSBD30-2 used for amplification of the soybeanBD30 3′ transcription terminator.

SEQ ID NO:95 is the complete sequence of plasmid pKR585 described inExample 13 and FIG. 6.

SEQ ID NO:96 is primer oCGR5-1 used for amplification of the M. alpinadelta-6 desaturase.

SEQ ID NO:97 is primer oCGR5-2 used for amplification of the M. alpinadelta-6 desaturase.

SEQ ID NO:98 is primer oSGly-1 used for amplification of the glycininGy1 promoter.

SEQ ID NO:99 is primer oSGly-2 used for amplification of the glycininGy1 promoter,

SEQ ID NO:100 is primer LegPro5′ used for amplification of the legA2promoter sequence.

SEQ ID NO:101 is primer LegPro3′ used for amplification of the legA2promoter sequence.

SEQ ID NO:102 is primer LegTerm5′ used for amplification of the leg2Atranscription terminator.

SEQ ID NO:103 is primer LegTerm3′ used for amplification of the leg2Atranscription terminator.

SEQ ID NO:104 is primer CGR4forward used for the amplification of the M.alpina desaturase.

SEQ ID NO:105 is primer CGR4reverse used for the amplification of the M.alpina desaturase.

SEQ ID NO:106 is the forward primer, RPB2forward, used for amplificationof the Mortierella alpine elongase.

SEQ ID NO:107 is the reverse primer, RPB2reverse, used for amplificationof the Mortierella alpine elongase.

SEQ ID NO:108 is primer Asc5 used to form the AscI liker.

SEQ ID NO:109 is primer Asc3 used to form the AscI liker.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and publications cited areincorporated herein by reference in their entirety.

This invention concerns the isolation and confirmation of the identityof a Fusarium moniliforme gene and a Magnaporthe grisea gene encoding aΔ15 desaturase and identified their orthologs in other fungi.Additionally, methods and compositions are provided which permitmodification of the long-chain polyunsaturated fatty acid (PUFA) contentand composition of plants, in particular, oilseed plants and oleaginousorganisms, including oleaginous yeast (e.g., Yarrowia lipolytica) andplants (e.g., soybean, corn and sunflower).

The invention relates to novel Δ15 desaturase enzymes and genes encodingthe same that may be used for the manipulation of biochemical pathwaysfor the production of healthful PUFAs. Thus, the subject invention findsmany applications. PUFAs, or derivatives thereof, made by themethodology disclosed herein can be used as dietary substitutes, orsupplements, particularly infant formulas, for patients undergoingintravenous feeding or for preventing or treating malnutrition.Alternatively, the purified PUFAs (or derivatives thereof) may beincorporated into cooking oils, fats or margarines formulated so that innormal use the recipient would receive the desired amount for dietarysupplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use (human or veterinary).In this case, the PUFAs are generally administered orally but can beadministered by any route by which they may be successfully absorbed,e.g., parenterally (e.g., subcutaneously, intramuscularly orintravenously), rectally, vaginally or topically (e.g., as a skinointment or lotion).

Supplementation of humans or animals with PUFAs produced by recombinantmeans can result in increased levels of the added PUFAs, as well astheir metabolic derivatives. For example, treatment with arachidonicacid (ARA) can result not only in increased levels of ARA, but alsodownstream products of ARA such as prostaglandins. Complex regulatorymechanisms can make it desirable to combine various PUFAs, or adddifferent conjugates of PUFAs, in order to prevent, control or overcomesuch mechanisms to achieve the desired levels of specific PUFAs in anindividual.

In the context of this disclosure, a number of terms shall be utilized.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

The term “Fusarium moniliforme” is synonymous with Fusariumverticilloides.

A “food analog” is a food-like product manufactured to resemble its foodcounterpart, whether meat, cheese, milk or the like, and is intended tohave the appearance, taste, and texture of its counterpart. Thus, theterm “food” as used herein also encompasses food analogs.

“Aquaculture feed” refers to feed used in aquafarming which concerns thepropagation, cultivation or farming of aquatic organisms, animals and/orplants in fresh or marine waters. The term “animal feed” as used hereinalso encompasses aquaculture feed.

The term “fatty acids” refers to long-chain aliphatic acids (alkanoicacids) of varying chain length, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of C atoms in the particular fatty acid and Y is the number ofdouble bonds.

Generally, fatty acids are classified as saturated or unsaturated. Theterm “saturated fatty acids” refers to those fatty acids that have no“double bonds” between their carbon backbone. In contrast, “unsaturatedfatty acids” have “double bonds” along their carbon backbones (which aremost commonly in the cis-configuration). “Monounsaturated fatty acids”have only one “double bond” along the carbon backbone (e.g., usuallybetween the 9^(th) and 10^(th) carbon atom as for palmitoleic acid(16:1) and oleic acid (18:1)), while “polyunsaturated fatty acids” (or“PUFAs”) have at least two double bonds along the carbon backbone (e.g.,between the 9^(th) and 10^(th), and 12^(th) and 13^(th) carbon atoms forlinoleic acid (18:2); and between the 9^(th) and 10^(th), 12^(th) and13^(th), and 15^(th) and 16^(th) for α-linolenic acid (18:3)).

“PUFAs” can be classified into two major families (depending on theposition (n) of the first double bond nearest the methyl end of thefatty acid carbon chain). Thus, the “omega-6 fatty acids” (ω-6 or n6)have the first unsaturated double bond six carbon atoms from the omega(methyl) end of the molecule and additionally have a total of two ormore double bonds, with each subsequent unsaturation occurring 3additional carbon atoms toward the carboxyl end of the molecule. Incontrast, the “omega-3 fatty acids” (ω-3 or n-3) have the firstunsaturated double bond three carbon atoms away from the omega end ofthe molecule and additionally have a total of three or more doublebonds, with each subsequent unsaturation occurring 3 additional carbonatoms toward the carboxyl end of the molecule.

For the purposes of the present disclosure, the omega-reference systemwill be used to indicate the number of carbons, the number of doublebonds and the position of the double bond closest to the omega carbon(but in the table below, we use the carboxyl terminus system ie,6,9,12), counting from the omega carbon (which is numbered 1 for thispurpose). This nomenclature is shown below in Table 2, in the columntitled “Shorthand Notation”. The remainder of the Table summarizes thecommon names of ω-3 and ω-6 fatty acids, the abbreviations that will beused throughout the specification, and each compound's chemical name.TABLE 2 Nomenclature Of Polyunsaturated Fatty Acids Shorthand CommonName Abbreviation Chemical Name Notation Linoleic LAcis-9,12-octadecadienoic 18:2 ω-6 γ-Linolenic GLA cis-6, 9, 12- 18:3 ω-6octadecatrienoic Eicosadienoic EDA cis-11, 14-eicosadienoic 20:2 ω-6Dihomo γ- DGLA cis-8, 11, 14- 20:3 ω-6 Linolenic eicosatrienoicArachidonic ARA cis-5, 8, 11, 14- 20:4 ω-6 eicosatetraenoic α-LinolenicALA cis-9, 12, 15- 18:3 ω-3 octadecatrienoic Stearidonic STA cis-6, 9,12, 15- 18:4 ω-3 octadecatetraenoic Eicosatrienoic ETrA cis-11, 14, 17-20:3 ω-3 eicosatrienoic Eicosa- ETA cis-8, 11, 14, 17- 20:4 ω-3tetraenoic eicosatetraenoic Eicosa- EPA cis-5, 8, 11, 14, 17- 20:5 ω-3pentaenoic eicosapentaenoic Docosa- DPA cis-7, 10, 13, 16, 19- 22:5 ω-3pentaenoic docosapentaenoic Docosa- DHA cis-4, 7, 10, 13, 16, 19- 22:6ω-3 hexaenoic docosahexaenoic

Examples of an omega-3 fatty acid include, but are not limited to, thefollowing list of fatty acids where numbers in brackets indicate theposition of double bonds from the carboxy-terminus of the fatty acid:alpha-linolenic acid [ALA; 18:3(9,12,15)], stearidonic acid [STA;18:4(6,9,12,15)], eicosatetraenoic acid [ETA; 20:4(8,11,14,17)],eicosapentaenoic acid [EPA; 20:5(5,8,11,14,17)], docosapentaenoic acid[DPA; 22:5(7,10,13,16,19)] and docosahexaenoic acid [DHA;22:6(4,7,10,13,16,19)].

Simialrly, examples of an omega-6 fatty acid include, but are notlimited to, the following list of fatty acids where numbers in bracketsindicate the position of double bonds from the carboxy-terminus of thefatty acid: linoleic acid [LA; 18:2(9,12)], gamma-linolenic acid [GLA;18:3(6,9,12)], dihomo-gamma-linolenic acid [DGLA; 20:3(8,11,14)],arachidonic acid [ARA; 20:4(5,8,11,14)] and docosatetraenoic acid [DTA;22:4(7,10,13,16)].

The term “concentration” as applied to the concentration of anyindividual fatty acid is hereby given to mean the amount of theparticular fatty acid divided by the total amount of all of the fattyacids in a sample. The concentration of omega-3 fatty acids is definedas the amount of all omega-3 fatty acids (as defined above) divided bythe total amount of all of the fatty acids in a sample. Theconcentration of omega-6 fatty acids is defined as the amount of allomega-6 fatty acids (as defined above) divided by the total amount ofall of the fatty acids in a sample. The fatty acid concentration istypically expressed as a weight percent (wt. %-mass of individual fattyacid divided by mass of all fatty acids times 100%) or mole percent (mol%-mols of individual fatty acid divided by total mols of fatty acidstimes 100%).

The term “ratio of omega-3 to omega-6 fatty acids” or “omega-3 toomega-6 ratio” (n-3/n-6) is hereby defined as the concentration ofomega-3 fatty acids divided by the concentration of omega-6 fatty acids(wt. % omega-3/wt. % omega-6 or mol % omega-3/mol % omega-6).

The term “essential fatty acid” refers to a particular PUFA that anindividual must ingest in order to survive, being unable to synthesizethe particular essential fatty acid de novo. Linoleic (18:2, ω-6) andlinolenic (18:3, ω-3) fatty acids are “essential fatty acids”, sincehumans cannot synthesize them and have to obtain them in their diet.

The term “fat” refers to a lipid substance that is solid at 25° C. andusually saturated.

The term “oil” refers to a lipid substance that is liquid at 25° C. andusually polyunsaturated. PUFAs are found in the oils of some algae,oleaginous yeast and filamentous fungi. “Microbial oils” or “single celloils” are those oils naturally produced by microorganisms during theirlifespan. Such oils can contain long-chain PUFAs.

The term “PUFA biosynthetic pathway enzyme” refers to any of thefollowing enzymes (and genes which encode said enzymes) associated withthe biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase,a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, aΔ9 desaturase, a Δ8 desaturase and/or an elongase(s).

The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set ofgenes which, when expressed under the appropriate conditions encodeenzymes that catalyze the production of either or both ω-3 and ω-6 fattyacids. Typically the genes involved in the ω-3/ω-6 fatty acidbiosynthetic pathway encode some or all of the following enzymes: Δ12desaturase, Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase, Δ15desaturase, Δ9 desaturase, Δ8 desaturase and Δ4 desaturase. Arepresentative pathway is illustrated in FIG. 2, providing for theconversion of oleic acid through various intermediates to DHA, whichdemonstrates how both ω-3 and ω-6 fatty acids may be produced from acommon source. The pathway is naturally divided into two portions whereone portion will generate ω-3 fatty acids and the other portion, onlyω-6 fatty acids. That portion that only generates ω-3 fatty acids willbe referred to herein as the ω-3 fatty acid biosynthetic pathway,whereas that portion that generates only ω-6 fatty acids will bereferred to herein as the ω-6 fatty acid biosynthetic pathway.

In humans there is evidence showing a lowering effect of ω-3 fatty acidson blood triacylglycerol levels. Other evidence supports a protectiverole against suffering a fatal heart attack. Both linoleic andα-linolenic acids are precursors for the synthesis of the eicosonoidsderived from their longer chain metabolites. During synthesis thesemetabolites compete for the same enzymes. Those derived from α-linolenicacid ω-3) tend to have less potent inflammatory and immunologicaleffects than those derived from linoleic acid (ω-6). Alpha-linolenicacid also gives rise to docosahexaenoic acid (DHA), a major constituentof the human brain and retina. The richest sources of alpha-linolenicacid are some seed oils, such as linseed oil, rapeseed oil, soya oil andsome nuts, particularly walnuts. The very long chain ω-3 fatty acids DHAand eicosapentaenoic acid (EPA), which can be made in the body fromalpha-linolenic acid, are provided in fish oils and the flesh ofoil-rich fish (not tinned tuna). Oils from flax, such as linseed oilthat is rich in α-linolenic acid, also have industrial applications as“drying oils” for use in varnishes and paints.

The term “functional” as used herein in context with the ω-3/ω-6 fattyacid biosynthetic pathway means that some (or all of) the genes in thepathway express active enzymes. It should be understood that “ω-3/ω-6fatty acid biosynthetic pathway” or “functional ω-3/ω-6 fatty acidbiosynthetic pathway” does not imply that all the genes listed in theabove paragraph are required, as a number of fatty acid products willonly require the expression of a subset of the genes of this pathway.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a mono-or polyunsaturated fatty acid or precursor which is of interest. Despiteuse of the omega-reference system throughout the specification inreference to specific fatty acids, it is more convenient to indicate theactivity of a desaturase by counting from the carboxyl end of thesubstrate using the Δ-system. Of particular interest herein are Δ15desaturases that desaturate a fatty acid between the 15^(th) and 16^(th)carbon atoms numbered from the carboxyl-terminal end of the molecule andthat catalyze the conversion of LA to ALA. Other desaturases relevant tothe present disclosure include: Δ12 desaturases that catalyze theconversion of oleic acid to LA; Δ17 desaturases that catalyze theconversion of DGLA to ETA and/or ARA to EPA; Δ6 desaturases thatcatalyze the conversion of LA to GLA and/or ALA to STA; Δ5 desaturasesthat catalyze the conversion of DGLA to ARA and/or ETA to EPA; Δ4desaturases that catalyze the conversion of DPA to DHA; Δ8 desaturasesthat catalyze the conversion of EDA to DGLA and/or ETrA to ETA; and Δ9desaturases that catalyze the conversion of palmitate to palmitoleicacid (16:1) and/or stearate to oleic acid (18:1). In the art, Δ15 andΔ17 desaturases are also occassionally referred to as “omega-3desaturases”, “w-3 desaturases”, and/or “ω-3 desaturases”. Somedesaturases have activities on two or more substrates (e.g., thesubstrates of the Saprolegnia diclina Δ17 desaturase include ARA andDGLA, those of the Caenorhabditis elegans ω-3 desaturase include LA andGLA, and those of the Fusarium moniliforme Δ-15 desaturase describedherein include LA, GLA and DGLA).

The term “proteins having homology to the Yarrowia lipolyticaΔ-12desaturase” refers to the proteins identified herein as SEQ ID NOs:2, 4,6, 8, 10, 12, 14,16,18 and 20, and that have homology to the Y.Iipolytica desaturase identified herein as SEQ ID NO:55 (characterizedin co-pending U.S. patent application Ser. No. 10/840325, hereinincorporated by reference in its entirety). Phylogenetic analysisdetermined that these proteins (i.e., SEQ ID NOs:2, 4, 6, 8,10,12,14,16,18 and 20) clustered into two distinct sub-families,referred to herein as “Sub-family 1” and “Sub-family 2”. Specifically,the Sub-family 1 proteins (i.e., SEQ ID NOs:2, 6, 10, 14 and 18) appearto encode Δ-15 desaturases as characterized herein. In contrast, theSub-family 2 proteins encode proteins with Δ-12 desaturase activity(i.e., SEQ ID NOs:4, 8,12,16 and 20; see co-pending U.S. ProvisionalApplication 60/570679, herein incorporated by reference in itsentirety).

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., a desaturaseor elongase) can convert substrate to product. The conversion efficiencyis measured according to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it. Inthe present Application, it is desirable to identify those Δ-15desaturases characterized by a high percent substrate conversion(([18:3]/[18:2+18:3])* 100) when expressed in eukaryotic organisms, suchas oleaginous yeast hosts; thus, for example, a conversion efficiency toALA of at least about 50% is useful, a conversion efficiency to ALA ofat least about 80% is preferred, while a conversion efficiency to ALA ofat least about 90% is particularly suitable, and a conversion efficiencyto ALA of at least about 95% is most preferred.

The term “elongase” refers to a polypeptide that can elongate a fattyacid carbon chain to produce an acid that is 2 carbons longer than thefatty acid substrate that the elongase acts upon. This process ofelongation occurs in a multi-step mechanism in association with fattyacid synthase, whereby CoA is the acyl carrier (Lassner et al., ThePlant Cell 8:281-292 (1996)). Briefly, malonyl-CoA is condensed with along-chain acyl-CoA to yield CO₂ and a β-ketoacyl-CoA (where the acylmoiety has been elongated by two carbon atoms). Subsequent reactionsinclude reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA, anda second reduction to yield the elongated acyl-CoA. Examples ofreactions catalyzed by elongases are the conversion of GLA to DGLA, STAto ETA, and EPA to DPA. Accordingly, elongases can have differentspecificities. For example, a C_(16/18) elongase will prefer a C₁₆substrate, a C_(18/20) elongase will prefer a C₁₈ substrate and aC_(20/22) elongase will prefer a C₂₀ substrate. In like manner, a Δ-9elongase is able to catalyze the conversion of LA and ALA to EDA andETrA, respectively.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2^(nd) ed., Plenum, 1980). These include oilseed plants(e.g., soybean, corn, safflower, sunflower, canola, rapeseed, flax,maize and primrose) and microorganisms (e.g., Thraustochytrium sp.,Schizochytrium sp., Mortierella sp. and certain oleaginous yeast).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can make oil. Generally, the cellular oil or triacylglycerolcontent of oleaginous microorganisms follows a sigmoid curve, whereinthe concentration of lipid increases until it reaches a maximum at thelate logarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol. 57:419-25 (1991)). Examples ofoleaginous yeast include, but are no means limited to, the followinggenera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus,Trichosporon and Lipomyces.

The term “fermentable carbon substrate” means a carbon source that amicroorganism will metabolize to derive energy. Typical carbonsubstrates of the invention include, but are not limited to:monosaccharides, oligosaccharides, polysaccharides, alkanes, fattyacids, esters of fatty acids, monoglycerides, carbon dioxide, methanol,formaldehyde, formate and carbon-containing amines.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid fragments for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid fragments to reflect the typical codon usage of the host organismwithout altering the polypeptide for which the DNA codes.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA. The terms“polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”,and “nucleic acid fragment”/“isolated nucleic acid fragment” are usedinterchangeably herein. These terms encompass nucleotide sequences andthe like. A polynucleotide may be a polymer of RNA or DNA that issingle- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by a single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant. Chimeric genes can be designed for use insuppression by linking a nucleic acid fragment or subfragment thereof,whether or not it encodes an active enzyme, in the sense or antisenseorientation relative to a plant promoter sequence.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid fragment can anneal to theother nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washedwith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)).In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence. The instantspecification teaches the complete amino acid and nucleotide sequenceencoding particular fungal proteins. The skilled artisan, having thebenefit of the sequences as reported herein, may now use all or asubstantial portion of the disclosed sequences for purposes known tothose skilled in this art. Accordingly, the instant invention comprisesthe complete sequences as reported in the accompanying Sequence Listing,as well as substantial portions of those sequences as defined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listing,as well as those substantially similar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: N.Y. (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: N.Y. (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: N.J. (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: N.Y. (1991). Preferred methods to determine identity aredesigned to give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences is performed using the Clustal method ofalignment (Higgins and Sharp, CABIOS. 5:151153 (1989)) with defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10), unless otherwisespecified. Default parameters for pairwise alignments using the Clustalmethod are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

Suitable nucleic acid fragments (isolated polynucleotides of the presentinvention) encode polypeptides that are at least about 70% identical,preferably at least about 75% identical, and more preferably at leastabout 80% identical to the amino acid sequences reported herein.Preferred nucleic acid fragments encode amino acid sequences that areabout 85% identical to the amino acid sequences reported herein. Morepreferred nucleic acid fragments encode amino acid sequences that are atleast about 90% identical to the amino acid sequences reported herein.Most preferred are nucleic acid fragments that encode amino acidsequences that are at least about 95% identical to the amino acidsequences reported herein. Suitable nucleic acid fragments not only havethe above homologies but typically encode a polypeptide having at least50 amino acids, preferably at least 100 amino acids, more preferably atleast 150 amino acids, still more preferably at least 200 amino acids,and most preferably at least 250 amino acids.

The term “homology” refers to the relationship among sequences wherebythere is some extent of likeness, typically due to descent from a commonancestral sequence. Homologous sequences can share homology based ongenic, structural, functional and/or behavioral properties. The term“ortholog” or “orthologous sequences” refers herein to a relationshipwhere sequence divergence follows speciation (i.e., homologous sequencesin different species arose from a common ancestral gene duringspeciation). In contrast, the term “paralogous” refers to homologoussequences within a single species that arose by gene duplication. Oneskilled in the art will be familiar with techniques required to identifyhomologous, orthologous and paralogous sequences.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize, under moderately stringent conditions (forexample, 0.5 ×SSC, 0.1% SDS, 60° C.) with the sequences exemplifiedherein, or to any portion of the nucleotide sequences disclosed hereinand which are functionally equivalent to any of the nucleic acidsequences disclosed herein. Stringency conditions can be adjusted toscreen for moderately similar fragments, such as homologous sequencesfrom distantly related organisms, to highly similar fragments, such asgenes that duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions involves a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDSat 50° C. for 30 min. A more preferred set of stringent conditionsinvolves the use of higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions involves the use of twofinal washes in 0.1×SSC, 0.1% SDS at 65° C.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Chemically synthesized”, as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures;or automated chemical synthesis can be performed using one of a numberof commercially available machines. “Synthetic genes” can be assembledfrom oligonucleotide building blocks that are chemically synthesizedusing procedures known to those skilled in the art. These buildingblocks are ligated and annealed to form gene segments that are thenenzymatically assembled to construct the entire gene. Accordingly, thegenes can be tailored for optimal gene expression based on optimizationof nucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell, where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and that may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene that is introduced intothe host organism by gene transfer. Foreign genes can comprise nativegenes introduced into a non-native organism, native genes introducedinto a new location within the native host, or chimeric genes. A“transgene” is a gene that has been introduced into the genome by atransformation procedure. A “codon-optimized gene” is a gene having itsfrequency of codon usage designed to mimic the frequency of preferredcodon usage of the host cell. An “allele” is one of several alternativeforms of a gene occupying a given locus on a chromosome. When all thealleles present at a given locus on a chromosome are the same that plantis homozygous at that locus. If the alleles present at a given locus ona chromosome differ that plant is heterozygous at that locus.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity. Promoters that cause agene to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro, J. K., and Goldberg, R. B.(1989) Biochemistry of Plants 15:1-82.

The “translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995) Mol.Biotechnol. 3:225-236).

The terms “3′ non-coding sequences” and “transcription terminator” referto DNA sequences located downstream of a coding sequence. This includespolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The 3′ region can influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” or “mRNA” refersto the RNA that is without introns and that can be translated intoprotein by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to, and derived from, mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO99/28508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated and yethas an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation. In another example, complementary RNA regions can beoperably linked, either directly or indirectly, 5′ to the target mRNA,or 3′ to the target mRNA, or within the target mRNA, or a firstcomplementary region is 5′ and its complement is 3′ to the target mRNA.

The term “expression”, as used herein, refers to the production of afunctional end-product e.g., a mRNA or a protein (precursor or mature).

The term “expression cassette” as used herein, refers to a discretenucleic acid fragment into which a nucleic acid sequence or fragment canbe moved.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

“Transformation” refers to the transfer of a nucleic acid fragment intoa host organism, resulting in genetically stable inheritance. Thenucleic acid fragment may be a plasmid that replicates autonomously, forexample; or, it may integrate into the genome of the host organism. Hostorganisms containing the transformed nucleic acid fragments are referredto as “transgenic” or “recombinant” or “transformed” organisms.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence (seeVaucheret et al. (1998) Plant J. 16:651-659; and Gura (2000) Nature404:804-808). The overall efficiency of this phenomenon is low, and theextent of the RNA reduction is widely variable. Recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication WO 99/53050 published on Oct. 21, 1999 and more recently,Applicants′ assignee's PCT Application having international publicationnumber WO 02/00904 published on Jan. 3, 2002). This increases thefrequency of co-suppression in the recovered transgenic plants. Anothervariation describes the use of plant viral sequences to direct thesuppression, or “silencing”, of proximal mRNA encoding sequences (PCTPublication WO 98/36083 published on Aug. 20, 1998). Both of theseco-suppressing phenomena have not been elucidated mechanistically,although genetic evidence has begun to unravel this complex situation(Elmayan et al. (1998) Plant Cell 10:1747-1757).

The polynucleotide sequences used for suppression do not necessarilyhave to be 100% complementary to the polynucleotide sequences found inthe gene to be suppressed. For example, suppression of all the subunitsof the soybean seed storage protein β-conglycinin has been accomplishedusing a polynucleotide derived from a portion of the gene encoding the asubunit (U.S. Pat. No. 6,362,399). β-conglycinin is a heterogeneousglycoprotein composed of varying combinations of three highly negativelycharged subunits identified as α, α′ and β. The polynucleotide sequencesencoding the α and α′ subunits are 85% identical to each other while thepolynucleotide sequences encoding the β subunit are 75 to 80% identicalto the a and a′ subunits. Thus, polynucleotides that are at least 75%identical to a region of the polynucleotide that is target forsuppression have been shown to be effective in suppressing the desiredtarget. The polynucleotide should be at least 80% identical, preferablyat least 90% identical, most preferably at least 95% identical, or thepolynucleotide may be 100% identical to the desired target.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing e.g., a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell.

“Transformation cassette” refers to a specific vector containing aforeign gene and having elements in addition to the foreign gene thatfacilitate transformation of a particular host cell. “Expressioncassette” refers to a specific vector containing a foreign gene andhaving elements in addition to the foreign gene that allow for enhancedexpression of that gene in a foreign host.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The terms “recombinant construct”, “expression construct”, “chimericconstruct”, “construct”, and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, a chimericconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such construct may be used byitself or may be used in conjunction with a vector. If a vector is usedthen the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al.,(1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.

The term “altered biological activity” will refer to an activity,associated with a protein encoded by a nucleotide sequence which can bemeasured by an assay method, where that activity is either greater thanor less than the activity associated with the native sequence. “Enhancedbiological activity” refers to an altered activity that is greater thanthat associated with the native sequence. “Diminished biologicalactivity” is an altered activity that is less than that associated withthe native sequence.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules (during cross over). The fragmentsthat are exchanged are flanked by sites of identical nucleotidesequences between the two DNA molecules (i.e., “regions of homology”).The term “regions of homology” refer to stretches of nucleotide sequenceon nucleic acid fragments that participate in homologous recombinationthat have homology to each other. Effective homologous recombinationwill take place where these regions of homology are at least about 10 bpin length where at least about 50 bp in length is preferred. Typicallyfragments that are intended for recombination contain at least tworegions of homology where targeted gene disruption or replacement isdesired.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist,L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a cycle.

Microbial Biosynthesis of Fatty Acids

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium (FIG. 1). When cells have exhausted available nitrogensupplies (e.g., when the carbon to nitrogen ratio is greater than about40), the depletion of cellular adenosine monophosphate (AMP) leads tothe cessation of AMP-dependent isocitrate dehydrogenase activity in themitochondria and the accumulation of citrate, transport of citrate intothe cytosol, and subsequent cleavage of the citrate by ATP-citrate lyaseto yield acetyl-CoA. Acetyl-CoA is the principle building block for denovo biosynthesis of fatty acids. Although any compound that caneffectively be metabolized to produce acetyl-CoA can serve as aprecursor of fatty acids, glucose is the primary source of carbon inthis type of reaction (FIG. 1). Glucose is converted to pyruvate viaglycolysis, and pyruvate is then transported into the mitochondria whereit can be converted to acetyl-CoA by pyruvate dehydrogenase (“PD”).Since acetyl-CoA can not be transported directly across themitochondrial membrane into the cytoplasm, the two carbons fromacetyl-CoA condense with oxaloacetate to yield citrate (catalyzed bycitrate synthase). Citrate is transported directly into the cytoplasm,where it is cleaved by ATP-citrate lyase to regenerate acetyl-CoA andoxaloacetate. The oxaloacetate reenters the tricarboxylic acid cycle,via conversion to malate.

The synthesis of malonyl-CoA is the first committed step of fatty acidbiosynthesis, which takes place in the cytoplasm. Malonyl-CoA isproduced via carboxylation of acetyl-CoA by acetyl-CoA carboxylase(“ACC”). Fatty acid synthesis is catalyzed by a multi-enzyme fatty acidsynthase complex (“FAS”) and occurs by the condensation of eighttwo-carbon fragments (acetyl groups from acetyl-CoA) to form a 16-carbonsaturated fatty acid, palmitate. More specifically, FAS catalyzes aseries of 7 reactions, which involve the following (Smith, S. FASEB J,8(15):1248-59 (1994)):

-   -   1. Acetyl-CoA and malonyl-CoA are transferred to the acyl        carrier protein (ACP) of FAS. The acetyl group is then        transferred to the malonyl group, forming β-ketobutyryl-ACP and        releasing CO₂.    -   2. The β-ketobutyryl-ACP undergoes reduction (via β-ketoacyl        reductase) and dehydration (via β-hydroxyacyl dehydratase) to        form a trans-monounsaturated fatty acyl group.    -   3. The double bond is reduced by NADPH, yielding a saturated        fatty-acyl group two carbons longer than the initial one. The        butyryl-group's ability to condense with a new malonyl group and        repeat the elongation process is then regenerated.    -   4. When the fatty acyl group becomes 16 carbons long, a        thioesterase activity hydrolyses it, releasing free palmitate.

Palmitate (16:0) is the precursor of longer chain saturated andunsaturated fatty acids (e.g., stearic (18:0), palmitoleic (16:1) andoleic (18:1) acids) through the action of elongases and desaturasespresent in the endoplasmic reticulum membrane. Palmitate and stearate(as CoA and/or ACP esters) are converted to their unsaturatedderivatives, palmitoleic (16:1) and oleic (18:1) acids, respectively, bythe action of a Δ-9 desaturase.

Triacylglycerols (the primary storage unit for fatty acids) are formedby the esterification of two molecules of acyl-CoA toglycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonlyidentified as phosphatidic acid) (FIG. 1). The phosphate is thenremoved, by phosphatidic acid phosphatase, to yield 1,2-diacylglycerol.Triacylglycerol is formed upon the addition of a third fatty acid, forexample, by the action of a diacylglycerol-acyl transferase.

Biosynthesis of Omega Fatty Acids

Simplistically, the metabolic process that converts LA to GLA, DGLA andARA (the ω-6 pathway) and ALA to STA, ETA, EPA, DPA and DHA (the ω-3pathway) involves elongation of the carbon chain through the addition oftwo-carbon units and desaturation of the molecule through the additionof double bonds (FIG. 2). This requires a series of special desaturationand elongation enzymes present in the endoplasmic reticulum membrane.

ω-6 Fatty Acids

Oleic acid is converted to LA (18:2), the first of the ω-6 fatty acids,by the action of a Δ-12 desaturase. Subsequent ω-6 fatty acids areproduced as follows: 1.) LA is converted to GLA by the action of a Δ-6desaturase; 2.) GLA is converted to DGLA by the action of an elongase;and 3.) DGLA is converted to ARA by the action of a Δ-5 desaturase.

Omega-3 Fatty Acids

Linoleic acid (LA) is converted to ALA, the first of the ω-3 fattyacids, by the action of a Δ-15 desaturase. Subsequent ω-3 fatty acidsare produced in a series of steps similar to that for the ω-6 fattyacids. Specifically: 1.) ALA is converted to STA by the activity of a Δ6desaturase; 2.) STA is converted to ETA by the activity of an elongase;and 3.) ETA is converted to EPA by the activity of a Δ5 desaturase.Alternatively, ETA and EPA can be produced from DGLA and ARA,respectively, by the activity of a Δ17 desaturase. EPA can be furtherconverted to DHA by the activity of an elongase and a Δ4 desaturase.

In alternate embodiments, a Δ9 elongase is able to catalyze theconversion of LA and ALA to EDA and ETrA, respectively. A Δ8 desaturasethen converts these products to DGLA and ETA, respectively.

Genes Involved in Omega Fatty Acid Production

Many microorganisms, including algae, bacteria, molds and yeast, cansynthesize PUFAs and omega fatty acids in the ordinary course ofcellular metabolism. Particularly well-studied are fungi includingSchizochytrium aggregatm, species of the genus Thraustochytrium andMorteriella alpina. Additionally, many dinoflagellates (Dinophyceaae)naturally produce high concentrations of PUFAs. As such, a variety ofgenes involved in oil production have been identified through geneticmeans and the DNA sequences of some of these genes are publiclyavailable (non-limiting examples are shown below in Table 3): TABLE 3Some Publicly Available Genes Involved In PUFA Production GenbankAccession No. Description AY131238 Argania spinosa Δ6 desaturase Y055118Echium pitardii var. pitardii Δ6 desaturase AY055117 Echium gentianoidesΔ6 desaturase AF296076 Mucor rouxii Δ6 desaturase AF007561 Boragoofficinalis Δ6 desaturase L11421 Synechocystis sp. Δ6 desaturaseNM_031344 Rattus norvegicus Δ6 fatty acid desaturase AF465283,Mortierella alpina Δ6 fatty acid desaturase AF465281, AF110510 AF465282Mortierella isabellina Δ6 fatty acid desaturase AF419296 Pythiumirregulare Δ6 fatty acid desaturase AB052086 Mucor circinelloides D6dmRNA for Δ6 fatty acid desaturase AJ250735 Ceratodon purpureus mRNA forΔ6 fatty acid desaturase AF126799 Homo sapiens Δ6 fatty acid desaturaseAF126798 Mus musculus Δ6 fatty acid desaturase AF199596, Homo sapiens Δ5desaturase AF226273 AF320509 Rattus norvegicus liver Δ5 desaturaseAB072976 Mus musculus D5D mRNA for Δ5 desaturase AF489588Thraustochytrium sp. ATCC21685 Δ5 fatty acid desaturase AJ510244Phytophthora megasperma mRNA for Δ5 fatty acid desaturase AF419297Pythium irregulare Δ5 fatty acid desaturase AF07879 Caenorhabditiselegans Δ5 fatty acid desaturase AF067654 Mortierella alpina Δ5 fattyacid desaturase AB022097 Dictyostelium discoideum mRNA for Δ5 fatty aciddesaturase AF489589.1 Thraustochytrium sp. ATCC21685 Δ4 fatty aciddesaturase AX464731 Mortierella alpina elongase gene (also WO 00/12720)AAG36933 Emericella nidulans oleate Δ2 desaturase AF110509, Mortierellaalpina Δ12 fatty acid AB020033 desaturase mRNA AAL13300 MortierellaalpinaΔ12 fatty acid desaturase AF417244 Mortierella alpina ATCC 16266Δ12 fatty acid desaturase gene AF161219 Mucor rouxii Δ12 desaturase mRNAX86736 Spiruline platensis Δ12 desaturase AF240777 Caenorhabditiselegans Δ12 desaturase AB007640 Chlamydomonas reinhardtii Δ12 desaturaseAB075526 Chlorella vulgaris Δ12 desaturase AP002063 Arabidopsis thalianamicrosomal Δ12 desaturase NP_441622, Synechocystis sp. PCC 6803 Δ15desaturase BAA18302, BAA02924 AAL36934 Perilla frutescens Δ15 desaturaseAF338466 Acheta domesticus Δ9 desaturase 3 mRNA AF438199 Picea glaucadesaturase Δ9 (Des9) mRNA E11368 Anabaena Δ9 desaturase E11367Synechocystis Δ9 desaturase D83185 Pichia angusta DNA for Δ9 fatty aciddesaturase U90417 Synechococcus vulcanus Δ9 acyl-lipid fatty aciddesaturase (desC) gene AF085500 Mortierella alpina Δ9desaturase mRNAAY504633 Emericella nidulans Δ9 stearic acid desaturase (sdeB) geneNM_069854 Caenorhabditis elegans essential fatty acid desaturase,stearoyl-CoA desaturase (39.1 kD) (fat-6) complete mRNA AF230693Brassica oleracea cultivar Rapid Cycling stearoyl-ACP desaturase(Δ9-BO-1) gene, exon sequence AX464731 Mortierella alpina elongase gene(also WO 02/08401) NM_119617 Arabidopsis thaliana fatty acid elongase 1(FAE1) (At4g34520) mRNA NM_134255 Mus musculus ELOVL family member 5,elongation of long chain fatty acids (yeast) (Elovl5), mRNA NM_134383Rattus norvegicus fatty acid elongase 2 (rELO2), mRNA NM_134382 Rattusnorvegicus fatty acid elongase 1 (rELO1), mRNA NM_068396, Caenorhabditiselegans fatty acid ELOngation NM_068392, (elo-6), (elo-5), (elo-2),(elo-3), NM_070713, and (elo-9) mRNA NM_068746, NM_064685

Additionally, the patent literature provides many additional DNAsequences of genes (and/or details concerning several of the genes aboveand their methods of isolation) involved in oil production. See, forexample: U.S. Pat. No. 5,968,809 (Δ6 desaturases); U.S. Pat. No.5,972,664 and U.S. Pat. No. 6,075,183 (Δ5 desaturases); WO 91/13972 andU.S. Pat. No. 5,057,419 (Δ9 desaturases); U.S. 2003/0196217 A1 (Δ17desaturases); WO 02/090493 (Δ4 desaturases); WO 94/11516, U.S. Pat. No.5,443,974, and U.S. patent application Ser. No. 10/840325 (Δ12desaturases); WO 00/12720 and U.S. 2002/0139974A1 (elongases). Each ofthese patents and applications are herein incorporated by reference intheir entirety.

Of particular interest herein are Δ15 desaturases, and morespecifically, Δ15 desaturases that are suitable for heterologousexpression in oleaginous yeast (e.g., Yarrowia lipolytica). Genesencoding Δ15 desaturase are known in the art; for example, they havepreviously been cloned from plants (e.g., Arabidopsis, Brassica napus,Glycine max (WO 93/11245)), cyanobacteria and C. elegans. Additionally,following the Applicants' invention described herein, fungal Δ15desaturases from Neurospora crassa, Botrytis cinerea and Aspergillusnidulans were disclosed in WO 03/099216 (published Dec. 4, 2003).

Many factors affect the choice of a specific polypeptide having Δ15desaturase activity that is to be expressed in a host cell forproduction of PUFAs (optionally in combination with other desaturasesand elongases). Depending upon the host cell, the availability ofsubstrate, and the desired end product(s), several polypeptides are ofinterest; however, considerations for choosing a specific polypeptidehaving desaturase activity include the substrate specificity of thepolypeptide, whether the polypeptide or a component thereof is arate-limiting enzyme, whether the desaturase is essential for synthesisof a desired PUFA, and/or co-factors required by the polypeptide. Theexpressed polypeptide preferably has parameters compatible with thebiochemical environment of its location in the host cell. For example,the polypeptide may have to compete for substrate with other enzymes inthe host cell. Analyses of the K_(M) and specific activity of thepolypeptide are therefore considered in determining the suitability of agiven polypeptide for modifying PUFA production in a given host cell.The polypeptide used in a particular host cell is one which can functionunder the biochemical conditions present in the intended host cell butotherwise can be any polypeptide having Δ15 desaturase activity capableof modifying the desired fatty acids (i.e., LA). Thus, the sequences maybe derived from any source, e.g., isolated from a natural source (frombacteria, algae, fungi, plants, animals, etc.), produced via asemi-synthetic route or synthesized de novo.

For the purposes of the present invention herein, however, it is usefulfor the polypeptide having Δ15 desaturase activity to have a conversionefficiency (i.e., ([18:3]/[18:2+18:3])* 100) of at least about 50% whenexpressed in the desired eukaryotic host cell, wherein a conversionefficiency of at least about 80% is more desirable, a conversionefficiency of at least about 90% is particularly suitable, and aconversion efficiency of at least about 95% is most preferred.

Identification of Novel Fungal Δ15 Desaturases

Several fungi, including the filamentous fungi Magnaporthe grisea,Neurospora crassa, Aspergillus nidulans, Fusarium graminearium andFusarium moniliforme are known to make 18:3 (WO 03/099216; WO 03/099216). In view of the teachings and discoveries disclosed herein each ofthese fungi are expected to have Δ15 desaturase enzyme activity. Thesesequences will be particularly for expression of the genes in oleaginousyeast (e.g., Yarrowia lipolytica).

A novel Δ15 desaturase from Fusarium moniliforme was identified herein,by sequence comparison using the Yarrowia lipolytica Δ12 desaturaseprotein sequence (SEQ ID NO:55) as a query sequence. Specifically, thisYarrowia query sequence was used to search putative encoded proteinsequences of a proprietary DuPont expressed sequence tag (EST) libraryof Fusarium moniliforme strain M-8114 (E.I. du Pont de Nemours and Co.,Inc., Wilmington, Del.). This resulted in the identification of twohomologous sequences, Fm1 (SEQ ID NO:2) and Fm2 (SEQ ID NO:4), encodedby the nucleotide sequences of SEQ ID NOs:1 and 3, respectively.

The Yarrowia Δ12 desaturase sequence was also used as a query againstpublic databases of several filamentous fungi; specifically, homologousprotein sequences were identified in Aspergillus nidulans (SEQ ID NOs:6and 8), Magnaporthe grisea (SEQ ID NOs:10 and 12), Neurospora crassa(SEQ ID NOs:14 and 16) and Fusarium graminearium (SEQ ID NOs:18 and 20).Subsequent phylogenetic and homology analysis, based on comparison ofthese sequences (i.e., SEQ ID NOs: 2, 4, 6, 8,10, 12, 14,16, 18 and 20)using the method of Clustal W (slow, accurate, Gonnet option; Thompsonet al. Nucleic Acids Res. 22:4673-4680 (1994)), revealed two distinct“sub-families” of proteins having homology with the Yarrowia Δ12desaturase. Specifically, all proteins of “sub-family 1” (SEQ ID NOs: 2,6, 10, 14 and 18) were at least 46.2% identical to each other and wereless than 39.6% identical to the proteins of “sub-family 2” (SEQ ID NOs:4, 8, 12, 16 and 20) (FIGS. 4 and 5; Clustal method of alignment(supra)). The proteins of sub-family 2 were at least 56.3 % identical toeach other.

Since Yarrowia is only able to synthesize 18:2 (but not 18:3) while eachof the filamentous fungi described above can make both 18:2 and ALA, andsince Yarrowia has a single Δ12 desaturase while each of the filamentousfungi had two homologs to the Yarrowia Δ12 desaturase, Applicantspostulated that one of the sub-families of desaturases in theseorganisms represented Δ12 desaturases and the other represented Δ15desaturases. This hypothesis was tested by determining the activity of arepresentative protein(s) within each of the two sub-families usingexpression analysis. Specifically, Fm2 was expressed in Yarrowialipolytica and found to encode a Δ12 desaturase (see co-pending U.S.Provisional Application 60/570679), while Fm1 and Mg1 were expressed inY. lipolytica as described herein and were characterized as Δ15desaturases (additionally having some Δ12 desaturase activity).

The Fusarium moniliforme Δ15 desaturase nucleotide and deduced aminoacid sequences (i.e., SEQ ID NOs:1 and 2, respectively) were compared topublic database sequences using a Blastp 2.2.5 program of alignment,with the following parameters: Expect value of 10, Matrix of BLOSUM62,and filter for low complexity (Altschul et al., Nucleic Acid Res.25(17):3389-3402 (1997)). Thus, the Fusarium moniliforme Δ15 desaturasenucleotide sequence was most similar to the Gibberella zeae PH-1sequence provided as GenBank Accession No. XM_(—)388066.1 (86% identicalover a length of 573 bp). GenBank Accession No. XM_(—)388066.1corresponds to the Gibberella zeae PH-1 protein of GenBank Accession No.XP_(—)388066.1 and SEQ ID NO:17 herein (i.e., the Fusarium gramineariumΔ15 desaturase ORF). Direct comparison reveals that the F. moniliformeand F. graminearium Δ15 desaturase ORFs are 87.4% identical over alength of 1211 bp.

Comparison of the Fusarium moniliforme Δ15 desaturase deduced amino acidsequence to public databases reveals that the most closely relatedsequence based on percent identity is GenBank Accession No.XM_(—)388066.1 (89% over the length of 193 amino acids). This is apartial amino acid sequence that corresponds to SEQ ID NO:18 herein,encoding the full length Fusarium graminearium Δ15 desaturase that is88.8% identical over its full length of 403 amino acids.

More preferred amino acid fragments are at least about 70%-80% identicalto the sequence herein, where those sequences that are 85%-90% identicalare particularly suitable and those sequences that are about 95%identical are most preferred. Similarly, preferred Δ15 desaturaseencoding nucleic acid sequences corresponding to the instant ORF arethose encoding active proteins and which are at least about 70%-80%identical to the nucleic acid sequence encoding the F. moniliforme Δ15desaturase reported herein, where those sequences that are 85%-90%identical are particularly suitable and those sequences that are about95% identical are most preferred. Useful percent identities forpracticing the present invention include, but are not limited to 45.4%,46.2%, 47%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.It is believed that any integer percentage between 46% and 100% would beuseful.

Identification and Isolation of Homologs

The Δ15 desaturase nucleic acid fragment of the instant invention may beused to identify and isolate genes encoding homologous proteins from thesame or other bacterial, algal, fungal or plant species.

Identification Techniques

For example, a substantial portion of the Fusarium moniliforme Δ15desaturase amino acid or nucleotide sequence described herein can beused to putatively identify related polypeptides or genes, either bymanual evaluation of the sequence by one skilled in the art, or bycomputer-automated sequence comparison and identification usingalgorithms such as BLAST (Basic Local Alignment Search Tool; Altschul,S. F., et al., J. Mol. Biol. 215:403410 (1993)) and ClustalW (Megalignprogram of DNASTAR software). As described above, use of the Yarrowialipolytica Δ12 desaturase (SEQ ID NO:55) permitted the identification ofa suite of fungal desaturases which, upon analysis, clustered as twodistinct sub-families of proteins (i.e., sub-family 1 and sub-family 2).Subfamily-1 comprised the Fusarium moniliforme Δ15 desaturase describedabove, as well as the proteins whose coding DNA sequences are foundwithin the following:

-   -   Contig 1.122 (scaffold 9) in the Aspergillus nidulans genome        project (sponsored by the Center for Genome Research (CGR),        Cambridge, Mass.) (SEQ ID NO:6);    -   Locus MG08474.1 in contig 2.1597 in the Magnaporthe grisea        genome project (sponsored by the CGR and International Rice        Blast Genome Consortium) (SEQ ID NO:10);    -   GenBank Accession No. MBX01000577 (Neurospora crassa) (SEQ ID        NO:14); and    -   Contig 1.320 in the Fusarium graminearium genome project        (sponsored by the CGR and the International Gibberella zeae        Genomics Consortium (IGGR)); BAA33772.1 (SEQ ID NO:18).        Each of the above proteins were hypothesized to encode a Δ15        desaturase. This hypothesis was confirmed for Aspergillus        nidulans and Neurospora crassa in WO 03/099216 and confirmed        herein for Magnaporthe grisea.

Analysis of the above proteins reveals that these proteins have at least46.2% sequence identity to the Fusarium monilifonne Δ15 desaturase (SEQID NO:2), according to the Clustal method of alignment (supra) (FIG. 5).Additionally, the Δ15 desaturases of sub-family 1 in the presentinvention were also compared to other known Δ15 desaturase proteins;however, the Δ15 desaturases of sub-family 1 herein are more homologousto the proteins of sub-family 2 (39.6% identity) than they are to anyother known Δ15 desaturases. One skilled in the art would be able to usesimilar methodology to identify other orthologous proteins that wouldalso cluster within sub-family 1 (identified herein as Δ15 desaturases).

Alternatively, any of the instant desaturase sequences (i.e., SEQ IDNOs:1, 2, 5, 6, 9, 10, 13, 14, 17, 18) may be employed as hybridizationreagents for the identification of homologs. The basic components of anucleic acid hybridization test include a probe, a sample suspected ofcontaining the gene or gene fragment of interest and a specifichybridization method. Probes of the present invention are typicallysingle-stranded nucleic acid sequences that are complementary to thenucleic acid sequences to be detected. Probes are “hybridizable” to thenucleic acid sequence to be detected. The probe length can vary from 5bases to tens of thousands of bases, and will depend upon the specifictest to be done. Typically a probe length of about 15 bases to about 30bases is suitable. Only part of the probe molecule need be complementaryto the nucleic acid sequence to be detected. In addition, thecomplementarity between the probe and the target sequence need not beperfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness and Chen, Nucl. Acids Res.19:5143-5151 (1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide, and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

Isolation Methods

The Fusarium moniliforme Δ15 desaturase nucleic acid fragment of theinstant invention (or any of the Δ15 desaturases identified herein [SEQID NOs:5, 6, 9, 10, 13, 14, 17 and 18]) may be used to isolate genesencoding homologous proteins from the same or other bacterial, algal,fungal or plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to: 1.)methods of nucleic acid hybridization; 2.) methods of DNA and RNAamplification, as exemplified by various uses of nucleic acidamplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or stranddisplacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and 3.) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to thedesaturases described herein could be isolated directly by using all ora portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired yeast or fungus usingmethodology well known to those skilled in the art (wherein those yeastor fungus producing ALA [or ALA-derivatives] would be preferred).Specific oligonucleotide probes based upon the instant nucleic acidsequences can be designed and synthesized by methods known in the art(Maniatis, supra). Moreover, the entire sequences can be used directlyto synthesize DNA probes by methods known to the skilled artisan (e.g.,random primers DNA labeling, nick translation or end-labelingtechniques), or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of (or full-length of) the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length DNA fragments under conditions ofappropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used inpolymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding microbial genes. Alternatively, thesecond primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs byusing PCR to amplify copies of the region between a single point in thetranscript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (Gibco/BRL,Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated(Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217(1989)).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of DNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen DNA expression libraries toisolate full-length DNA clones of interest (Lerner, R. A. Adv. Immunol.36:1 (1984); Maniatis, supra).

Gene Optimization for Improved Heterologous Expression

A variety of techniques can be utilized to improve the expression of aparticular Δ15 desaturase of interest in an alternative host. Two suchtechniques include codon-optimization and mutagenesis of the gene.

Codon Optimization

For some embodiments, it may be desirable to modify a portion of thecodons encoding polypeptides having Δ15 desaturase activity, forexample, to enhance the expression of genes encoding those polypeptidesin an alternative host (i.e., oleaginous yeast).

In general, host preferred codons can be determined within a particularhost species of interest by examining codon usage in proteins(preferably those proteins expressed in the largest amount) anddetermining which codons are used with highest frequency. Then, thecoding sequence for the polypeptide of interest having desaturaseactivity can be synthesized in whole or in part using the codonspreferred in the host species. All (or portions) of the DNA also can besynthesized to remove any destabilizing sequences or regions ofsecondary structure that would be present in the transcribed mRNA. All(or portions) of the DNA also can be synthesized to alter the basecomposition to one more preferable in the desired host cell.

In preferred embodiments of the invention, the Δ15 desaturases frome.g., Fusarium moniliforme, Aspergillus nidulans, Magnaporthe grisea,Neurospora crassa and Fusarium graminearium could be codon-optimizedprior to their expression in a heterologous oleaginous yeast host, suchas Yarrowia lipolytica.

Mutagenesis

Methods for synthesizing sequences and bringing sequences together arewell established in the literature. For example, in vitro mutagenesisand selection, site-directed mutagenesis, error prone PCR (Melnikov etal., Nucleic Acids Research, 27(4):1056-1062 (Feb. 15, 1999)), “geneshuffling” (U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat.No. 5,830,721; and U.S. Pat. No. 5,837,458) or other means can beemployed to obtain mutations of naturally occurring desaturase genes,such as the Δ15 desaturases described herein. This would permitproduction of a polypeptide having desaturase activity in vivo with moredesirable physical and kinetic parameters for function in the host cell(e.g., a longer half-life or a higher rate of production of a desiredPUFA).

If desired, the regions of a desaturase polypeptide important forenzymatic activity can be determined through routine mutagenesis,expression of the resulting mutant polypeptides and determination oftheir activities. Mutants may include deletions, insertions and pointmutations, or combinations thereof. A typical functional analysis beginswith deletion mutagenesis to determine the N— and C-terminal limits ofthe protein necessary for function, and then internal deletions,insertions or point mutants are made to further determine regionsnecessary for function. Other techniques such as cassette mutagenesis ortotal synthesis also can be used. Deletion mutagenesis is accomplished,for example, by using exonucleases to sequentially remove the 5′ or 3′coding regions. Kits are available for such techniques. After deletion,the coding region is completed by ligating oligonucleotides containingstart or stop codons to the deleted coding region after the 5′ or 3′deletion, respectively. Alternatively, oligonucleotides encoding startor stop codons are inserted into the coding region by a variety ofmethods including site-directed mutagenesis, mutagenic PCR or byligation onto DNA digested at existing restriction sites. Internaldeletions can similarly be made through a variety of methods includingthe use of existing restriction sites in the DNA, by use of mutagenicprimers via site-directed mutagenesis or mutagenic PCR. Insertions aremade through methods such as linker-scanning mutagenesis, site-directedmutagenesis or mutagenic PCR. Point mutations are made throughtechniques such as site-directed mutagenesis or mutagenic PCR.

Chemical mutagenesis also can be used for identifying regions of adesaturase polypeptide important for activity. A mutated construct isexpressed, and the ability of the resulting altered protein to functionas a desaturase is assayed. Such structure-function analysis candetermine which regions may be deleted, which regions tolerateinsertions, and which point mutations allow the mutant protein tofunction in substantially the same way as the native desaturase. Allsuch mutant proteins and nucleotide sequences encoding them that arederived from the desaturase genes described herein are within the scopeof the present invention.

Thus, the present invention comprises the complete sequences of the Δ15desaturase genes as reported in the accompanying Sequence Listing, thecomplement of those complete sequences, substantial portions of thosesequences, codon-optimized desaturases derived therefrom, and thosesequences that are substantially homologous thereto.

Microbial Production of ω-3 and/or ω-6 Fatty Acids

Microbial production of ω-3 and/or ω-6 fatty acids can have severaladvantages over purification from natural sources such as fish orplants. For example:

-   -   1.) Many microbes are known with greatly simplified oil        compositions compared with those of higher organisms, making        purification of desired components easier;    -   2.) Microbial production is not subject to fluctuations caused        by external variables, such as weather and food supply;    -   3.) Microbially produced oil is substantially free of        contamination by environmental pollutants;    -   4.) Microbes can provide PUFAs in particular forms which may        have specific uses; and    -   5.) Microbial oil production can be manipulated by controlling        culture conditions, notably by providing particular substrates        for microbially expressed enzymes, or by addition of compounds        or genetic engineering approaches to suppress undesired        biochemical pathways.        In addition to these advantages, production of ω-3 and/or ω-6        fatty acids from recombinant microbes provides the ability to        alter the naturally occurring microbial fatty acid profile by        providing new biosynthetic pathways in the host or by        suppressing undesired pathways, thereby increasing levels of        desired PUFAs (or conjugated forms thereof and decreasing levels        of undesired PUFAs (see co-pending U.S. patent application Ser.        No. 10/840579, herein incorporated entirely by reference).

Methods for Production of Various ω-3 and/or ω-6 Fatty Acids

It is expected that introduction of chimeric genes encoding the Δ15desaturases described herein, under the control of the appropriatepromoters will result in increased production of ALA in the transformedhost organism. As such, the present invention encompasses a method forthe direct production of PUFAs comprising exposing a fatty acidsubstrate (i.e., LA) to the PUFA enzyme(s) described herein (e.g., theFusarium moniliforme Δ15 desaturase), such that the substrate isconverted to the desired fatty acid product (i.e., ALA). Morespecifically, it is an object of the present invention to provide amethod for the production of ALA in a microorganism (e.g., oleaginousyeast), wherein the microorganism is provided:

-   -   (a) an isolated nucleic acid fragment encoding a fungal protein        having Δ15 desaturase activity that has at least 46.2% identity        based on the Clustal method of alignment when compared to a        polypeptide having the sequence as set forth in SEQ ID NO:2;        and,    -   (b) a source of desaturase substrate consisting of LA;

wherein the yeast is grown under conditions such that the chimericdesaturase gene is expressed and the LA is converted to ALA, and whereinthe ALA is optionally recovered. Thus, this method minimally includesthe use of the following Δ15 desaturases: SEQ ID NOs:2, 6, 10, 14 and18, as described herein.

Alternatively, each PUFA gene and its corresponding enzyme productdescribed herein can be used indirectly for the production of ω-3 PUFAs.Indirect production of ω-3 PUFAs occurs wherein the fatty acid substrateis converted indirectly into the desired fatty acid product, via meansof an intermediate step(s) or pathway intermediate(s). Thus, it iscontemplated that the Δ15 desaturases described herein may be expressedin conjunction with one or more genes that encode other enzymes, suchthat a series of reactions occur to produce a desired product. In apreferred embodiment, for example, a host organism may be co-transformedwith a vector comprising additional genes encoding enzymes of the PUFAbiosynthetic pathway to result in higher levels of production of ω-3fatty acids (e.g., ALA, STA, ETA, EPA, DPA and DHA). Specifically, forexample, it may be desirable to over-express any one of the Δ15desaturases described herein in host cells that are also expressing: 1.)a gene encoding a Δ12 desaturase for the overproduction of ALA (whereinproduction is increased relative to expression of the Δ15 desaturasealone); 2.) a gene encoding a Δ6 desaturase (and optionally a Δ12desaturase) for the overproduction of STA; 3.) genes encoding a Δ6desaturase and high-affinity elongase (and optionally a Δ12 desaturase)for the overproduction of ETA; and 4.) genes encoding a Δ6 desaturase,high-affinity elongase and Δ5 desaturase (and optionally a Δ12desaturase) for the overproduction of EPA. As is well known to oneskilled in the art, various other combinations of the followingenzymatic activities may be useful to express in a host in conjunctionwith the desaturase(s) herein: a Δ4 desaturase, a Δ5 desaturase, a Δ6desaturase, a Δ12 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8desaturase, and/or an elongase (see FIG. 2). The particular genesincluded within a particular expression cassette will depend on the hostcell (and its PUFA profile and/or desaturase profile), the availabilityof substrate and the desired end product(s).

In alternative embodiments, it may be useful to disrupt a hostorganism's native Δ15 desaturase, based on the complete sequencesdescribed herein, the complement of those complete sequences,substantial portions of those sequences, codon-optimized desaturasesderived therefrom and those sequences that are substantially homologousthereto. For example, the targeted disruption of the Δ15 desaturase in ahost organism produces a mutant strain that is unable to synthesize ALA.This mutant strain could be useful for the production of “pure” ω-6fatty acids (without co-synthesis of ω-3 fatty acids).

Expression Systems, Cassettes and Vectors

The genes and gene products of the instant sequences described hereinmay be expressed in heterologous microbial host cells, particularly inthe cells of oleaginous yeast (e.g., Yarrowia lipolytica). Expression inrecombinant microbial hosts may be useful for the production of variousPUFA pathway intermediates, or for the modulation of PUFA pathwaysalready existing in the host for the synthesis of new productsheretofore not possible using the host.

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the encoded enzymes.

Vectors or DNA cassettes useful for the transformation of suitable hostcells are well known in the art. The specific choice of sequencespresent in the construct is dependent upon the desired expressionproducts (supra), the nature of the host cell and the proposed means ofseparating transformed cells versus non-transformed cells. Typically,however, the vector or cassette contains sequences directingtranscription and translation of the relevant gene(s), a selectablemarker and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene thatcontrols transcriptional initiation and a region 3′ of the DNA fragmentthat controls transcriptional termination. It is most preferred whenboth control regions are derived from genes from the transformed hostcell, although it is to be understood that such control regions need notbe derived from the genes native to the specific species chosen as aproduction host.

Initiation control regions or promoters which are useful to driveexpression of the instant ORFs in the desired host cell are numerous andfamiliar to those skilled in the art. Virtually any promoter capable ofdirecting expression of these genes in the selected host cell issuitable for the present invention. Expression in a host cell can beaccomplished in a transient or stable fashion. Transient expression canbe accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest. Stable expression can beachieved by the use of a constitutive promoter operably linked to thegene of interest. As an example, when the host cell is yeast,transcriptional and translational regions functional in yeast cells areprovided, particularly from the host species. The transcriptionalinitiation regulatory regions can be obtained, for example, from: 1.)genes in the glycolytic pathway, such as alcohol dehydrogenase,glyceraldehyde-3-phosphate-dehydrogenase (see U.S. patent applicationSer. No. 10/869630), phosphoglycerate mutase (see U.S. patentapplication Ser. No. 10/869630), fructose-bisphosphate aldolase (seeU.S. Patent Application No. 60/519971), phosphoglucose-isomerase,phosphoglycerate kinase, glycerol-3-phosphate O-acyltransferase (seeU.S. Patent Application No. 60/610060), etc.; or, 2.) regulatable genessuch as acid phosphatase, lactase, metallothionein, glucoamylase, thetranslation elongation factor EF1-α (TEF) protein (U.S. Pat. No.6,265,185), ribosomal protein S7 (U.S. Pat. No. 6,265,185), etc. Any oneof a number of regulatory sequences can be used, depending upon whetherconstitutive or induced transcription is desired, the efficiency of thepromoter in expressing the ORF of interest, the ease of construction andthe like.

Nucleotide sequences surrounding the translational initiation codon ATGhave been found to affect expression in yeast cells. If any of theinstant Δ15 desaturases are poorly expressed in yeast, the nucleotidesequences of exogenous genes can be modified to include an efficientyeast translation initiation sequence to obtain optimal gene expression.For expression in yeast, this can be done by site-directed mutagenesisof an inefficiently expressed gene by fusing it in-frame to anendogenous yeast gene, preferably a highly expressed gene.Alternatively, one can determine the consensus translation initiationsequence in the host and engineer this sequence into heterologous genesfor their optimal expression in the host of interest (see, e.g., U.S.patent application Ser. No. 10/840478 for specific teachings applicablefor Yarrowia lipolytica).

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Preferably, thetermination region is derived from a yeast gene, particularlySaccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces.The 3′-regions of mammalian genes encoding γ-interferon and α-2interferon are also known to function in yeast. Termination controlregions may also be derived from various genes native to the preferredhosts. Optionally, a termination site may be unnecessary; however, it ismost preferred if included.

As one of skill in the art is aware, merely inserting a gene into acloning vector does not ensure that it will be successfully expressed atthe level needed. In response to the need for a high expression rate,many specialized expression vectors have been created by manipulating anumber of different genetic elements that control aspects oftranscription, translation, protein stability, oxygen limitation, andsecretion from the host cell. More specifically, some of the molecularfeatures that have been manipulated to control gene expression include:1.) the nature of the relevant transcriptional promoter and terminatorsequences; 2.) the number of copies of the cloned gene and whether thegene is plasmid-borne or integrated into the genome of the host cell;3.) the final cellular location of the synthesized foreign protein; 4.)the efficiency of translation in the host organism; 5.) the intrinsicstability of the cloned gene protein within the host cell; and 6.) thecodon usage within the cloned gene, such that its frequency approachesthe frequency of preferred codon usage of the host cell. Each of thesetypes of modifications are encompassed in the present invention, asmeans to further optimize expression of the Δ15 desaturases describedherein.

Transformation Of Microbial Hosts

Once the DNA encoding a polypeptide suitable for expression in anappropriate microbial host has been obtained, it is placed in a plasmidvector capable of autonomous replication in a host cell, or it isdirectly integrated into the genome of the host cell. Integration ofexpression cassettes can occur randomly within the host genome or can betargeted through the use of constructs containing regions of homologywith the host genome sufficient to target recombination with the hostlocus. Where constructs are targeted to an endogenous locus, all or someof the transcriptional and translational regulatory regions can beprovided by the endogenous locus.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other construct(s) to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct(s) can be experimentallydetermined so that all introduced genes are expressed at the necessarylevels to provide for synthesis of the desired products.

Constructs comprising the gene of interest may be introduced into a hostcell by any standard technique. These techniques include transformation(e.g., lithium acetate transformation [Methods in Enzymology,194:186-187 (1991)]), protoplast fusion, biolistic impact,electroporation, microinjection, or any other method that introduces thegene of interest into the host cell. More specific teachings applicablefor oleaginous yeast (i.e., Yarrowia lipolytica) include U.S. Pat. Nos.4,880,741 and 5,071,764 and Chen, D. C. et al. (Appl MicrobiolBiotechnol. 48(2):232-235 (1997)).

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed” or “recombinant” herein. The transformed host willhave at least one copy of the expression construct and may have two ormore, depending upon whether the gene is integrated into the genome,amplified, or is present on an extrachromosomal element having multiplecopy numbers. The transformed host cell can be identified by selectionfor a marker contained on the introduced construct. Alternatively, aseparate marker construct may be co-transformed with the desiredconstruct, as many transformation techniques introduce many DNAmolecules into host cells. Typically, transformed hosts are selected fortheir ability to grow on selective media. Selective media mayincorporate an antibiotic or lack a factor necessary for growth of theuntransformed host, such as a nutrient or growth factor. An introducedmarker gene may confer antibiotic resistance, or encode an essentialgrowth factor or enzyme, thereby permitting growth on selective mediawhen expressed in the transformed host. Selection of a transformed hostcan also occur when the expressed marker protein can be detected, eitherdirectly or indirectly. The marker protein may be expressed alone or asa fusion to another protein. The marker protein can be detected by: 1.)its enzymatic activity (e.g. β-galactosidase can convert the substrateX-gal [5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside] to a coloredproduct; luciferase can convert luciferin to a light-emitting product);or 2.) its light-producing or modifying characteristics (e.g., the greenfluorescent protein of Aequorea victoria fluoresces when illuminatedwith blue light). Alternatively, antibodies can be used to detect themarker protein or a molecular tag on, for example, a protein ofinterest. Cells expressing the marker protein or tag can be selected,for example, visually, or by techniques such as FACS or panning usingantibodies. For selection of yeast transformants, any marker thatfunctions in yeast may be used. Desirably, resistance to kanamycin,hygromycin and the amino glycoside G418 are of interest, as well asability to grow on media lacking uracil or leucine.

Following transformation, substrates suitable for the instant Δ15desaturases (and, optionally other PUFA enzymes that are co-expressedwithin the host cell) may be produced by the host either naturally ortransgenically, or they may be provided exogenously.

Metabolic Engineering of ω-3 and/or ω-6 Fatty Acid Biosynthesis inMicrobes

Knowledge of the sequences of the present Δ15 desaturases will be usefulfor manipulating ω-3 and/or ω-6 fatty acid biosynthesis in oleaginousyeast, and particularly, in Yarrowia lipolytica. This may requiremetabolic engineering directly within the PUFA biosynthetic pathway oradditional manipulation of pathways that contribute carbon to the PUFAbiosynthetic pathway. Methods useful for manipulating biochemicalpathways are well known to those skilled in the art.

Techniques to UP-Regulate Desirable Biosynthetic Pathways

Additional copies of desaturase (and optionally elongase) genes may beintroduced into the host to increase the output of the ω-3 and/or ω-6fatty acid biosynthesis pathways, typically through the use of multicopyplasmids. Expression of desaturase and elongase genes also can beincreased at the transcriptional level through the use of a strongerpromoter (either regulated or constitutive) to cause increasedexpression, by removing/deleting destabilizing sequences from either themRNA or the encoded protein, or by adding stabilizing sequences to themRNA (U.S. Pat. No. 4,910,141). Yet another approach to increaseexpression of heterologous desaturase or elongase genes is to increasethe translational efficiency of the encoded mRNAs by replacement ofcodons in the native gene with those for optimal gene expression in theselected host microorganism.

Techniques to Down-Regulate Undesirable Biosynthetic Pathways

Conversely, biochemical pathways competing with the ω-3 and/or ω-6 fattyacid biosynthesis pathways for energy or carbon, or native PUFAbiosynthetic pathway enzymes that interfer with production of aparticular PUFA end-product, may be eliminated by gene disruption ordown-regulated by other means (e.g., antisense mRNA). For genedisruption, a foreign DNA fragment (typically a selectable marker gene)is inserted into the structural gene to be disrupted in order tointerrupt its coding sequence and thereby functionally inactivate thegene. Transformation of the disruption cassette into the host cellresults in replacement of the functional native gene by homologousrecombination with the non-functional disrupted gene (see, for example:Hamilton et al. J. Bacteriol. 171:46174622 (1989); Balbas et al. Gene136:211-213 (1993); Gueldener et al. Nucleic Acids Res. 24:2519-2524(1996); and Smith et al. Methods Mol. Cell. Biol. 5:270-277(1996)).

Antisense technology is another method of down-regulating genes when thesequence of the target gene is known. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed.This construct is then introduced into the host cell and the antisensestrand of RNA is produced. Antisense RNA inhibits gene expression bypreventing the accumulation of mRNA that encodes the protein ofinterest. The person skilled in the art will know that specialconsiderations are associated with the use of antisense technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of antisense genes may require the use of differentchimeric genes utilizing different regulatory elements known to theskilled artisan.

Although targeted gene disruption and antisense technology offereffective means of down-regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence-based. For example, cells may be exposed to UV radiation andthen screened for the desired phenotype. Mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect nonreplicating DNA (e.g., HNO₂and NH₂OH), as well as agents that affect replicating DNA (e.g.,acridine dyes, notable for causing frameshift mutations). Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. See, for example: Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed. (1989)Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl.Biochem. Biotechnol., 36:227 (1992).

Another non-specific method of gene disruption is the use oftransposable elements or transposons. Transposons are genetic elementsthat insert randomly into DNA but can be later retrieved on the basis ofsequence to determine where the insertion has occurred. Both in vivo andin vitro transposition methods are known. Both methods involve the useof a transposable element in combination with a transposase enzyme. Whenthe transposable element or transposon is contacted with a nucleic acidfragment in the presence of the transposase, the transposable elementwill randomly insert into the nucleic acid fragment. The technique isuseful for random mutagenesis and for gene isolation, since thedisrupted gene may be identified on the basis of the sequence of thetransposable element. Kits for in vitro transposition are commerciallyavailable [see, for example: 1.) The Primer Island Transposition Kit,available from Perkin Elmer Applied Biosystems, Branchburg, N.J., basedupon the yeast Ty1 element; 2.) The Genome Priming System, availablefrom New England Biolabs, Beverly, Mass., based upon the bacterialtransposon Tn7; and 3.) the EZ::TN Transposon Insertion Systems,available from Epicentre Technologies, Madison, Wis., based upon the Tn5bacterial transposable element].

Within the context of the present invention, it may be useful tomodulate the expression of the fatty acid biosynthetic pathway by anyone of the methods described above. For example, the present inventionprovides genes (i.e., Δ15 desaturases) encoding key enzymes in thebiosynthetic pathways leading to the production of ω-3 and/or ω-6 fattyacids. It will be particularly useful to express these genes inoleaginous yeast that produce insufficient amounts of 18:3 fatty acidsand to modulate the expression of this and other PUFA biosynthetic genesto maximize production of preferred PUFA products using various meansfor metabolic engineering of the host organism. Likewise, to maximizePUFA production with these genes, it may be necessary to disruptpathways that compete for the carbon flux directed toward PUFAbiosynthesis. In alternate embodiments, it may be desirable to disruptthe Δ15 desaturase herein, to promote synthesis of ω-6 fatty acids whilesimultaneously preventing co-synthesis of ω-3 fatty acids. In anotheralternate embodiment it will be possible to regulate the production ofω-3 and/or ω-6 fatty acids by placing any of the present Δ15 desaturasegenes under the control of inducible or regulated promoters.

Preferred Hosts for Recombinant Expression of Δ15 Desaturases

Host cells for expression of the instant genes and nucleic acidfragments may include microbial hosts that grow on a variety offeedstocks, including simple or complex carbohydrates, organic acids andalcohols, and/or hydrocarbons over a wide range of temperature and pHvalues. Although the genes described in the instant invention have beenisolated for expression in an oleaginous yeast, and in particularYarrowia lipolytica, it is contemplated that because transcription,translation and the protein biosynthetic apparatus is highly conserved,any bacteria, yeast, algae and/or filamentous fungus will be a suitablehost for expression of the present nucleic acid fragments.

Preferred hosts are oleaginous organisms, such as oleaginous yeast.These oleaginous organisms are naturally capable of oil synthesis andaccumulation, wherein the oil can comprise greater than about 25% of thecellular dry weight, more preferably greater than about 30% of thecellular dry weight, and most preferably greater than about 40% of thecellular dry weight. Genera typically identified as oleaginous yeastinclude, but are not limited to: Yarrowia, Candida, Rhodotorula,Rhodospodidium, Cryptococcus, Trichosporon and Lipomyces. Morespecifically, illustrative oil-synthesizing yeast include: Rhodospoddiumtoruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C.pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T.cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica(formerly classified as Candida lipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in afurther embodiment, most preferred are the Yarrowia lipolytica strainsdesignated as ATCC #76982, ATCC #20362, ATCC #8862, ATCC #18944 and/orLGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol.82(1):43-9 (2002)).

Other preferred microbial hosts include oleaginous bacteria, algae andother fungi; and, within this group of microbial hosts, of particularinterest are microorganisms that synthesize ω-6 fatty acids such as GLAand ARA. Thus, for example, transformation of Mortierella alpina (whichis commercially used for production of ARA) with the any of the presentΔ15 desaturase genes under the control of inducible or regulatedpromoters could yield a transformant organism capable of synthesizingEPA. Furthermore, one could improve the ratio of ω-3 to ω-6 fatty acidsis this genetically engineered organism by transforming those strainshaving a disruption or mutation in their native Δ12 desaturase (e.g., byintroducing any of the present Δ15 desaturases into the locus of thenative Δ12 gene, using means well known in the art). The method oftransformation of M. alpina described by Mackenzie et al. (Applied andEnvironmental Microbiology 66:4655 (2000)).

Fermentation Processes for PUFA Production

The transformed microbial host cell is grown under conditions thatoptimize activity of fatty acid biosynthetic genes and produce thegreatest and the most economical yield of fatty acids (e.g., ALA, whichcan in turn increase the production of various ω-3 fatty acids). Ingeneral, media conditions which may be optimized include the type andamount of carbon source, the type and amount of nitrogen source, thecarbon-to-nitrogen ratio, the oxygen level, growth temperature, pH,length of the biomass production phase, length of the oil accumulationphase and the time of cell harvest. Microorganisms of interest, such asoleaginous yeast, are grown in complex media (e.g., yeastextract-peptone-dextrose broth (YPD)) or a defined minimal media thatlacks a component necessary for growth and thereby forces selection ofthe desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources may include, but are not limitedto: monosaccharides (e.g., glucose, fructose), disaccharides (e.g.,lactose or sucrose), oligosaccharides, polysaccharides (e.g., starch,cellulose or mixtures thereof, sugar alcohols (e.g., glycerol) ormixtures from renewable feedstocks (e.g., cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt). Additionally,carbon sources may include alkanes, fatty acids, esters of fatty acids,monoglycerides, diglycerides, triglycerides, phospholipids and variouscommercial sources of fatty acids including vegetable oils (e.g.,soybean oil) and animal fats. Additionally, the carbon substrate mayinclude one-carbon substrates (e.g., carbon dioxide or methanol) forwhich metabolic conversion into key biochemical intermediates has beendemonstrated. Hence it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing substrates and will only be limited by the choice ofthe host organism. Although all of the above mentioned carbon substratesand mixtures thereof are expected to be suitable in the presentinvention, preferred carbon substrates are sugars and/or fatty acids.Most preferred is glucose and/or fatty acids containing between 10-22carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organicsource (e.g., urea or glutamate). In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins, and other componentsknown to those skilled in the art, suitable for the growth of themicroorganism and promotion of the enzymatic pathways necessary for PUFAproduction. Particular attention is given to several metal ions (e.g.,Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids and PUFAs(Nakahara, T. et al. Ind. Appl. Single Cell Oils, D. J. Kyle and R.Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of the particularmicroorganism will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of PUFAs in oleaginous yeast. In this approach, the firststage of the fermentation is dedicated to the generation andaccumulation of cell mass and is characterized by rapid cell growth andcell division. In the second stage of the fermentation, it is preferableto establish conditions of nitrogen deprivation in the culture topromote high levels of lipid accumulation. The effect of this nitrogendeprivation is to reduce the effective concentration of AMP in thecells, thereby reducing the activity of the NAD-dependent isocitratedehydrogenase of mitochondria. When this occurs, citric acid willaccumulate, thus forming abundant pools of acetyl-CoA in the cytoplasmand priming fatty acid synthesis. Thus, this phase is characterized bythe cessation of cell division followed by the synthesis of fatty acidsand accumulation of oil.

Although cells are typically grown at about 30° C., some studies haveshown increased synthesis of unsaturated fatty acids at lowertemperatures (Yongmanitchai and Ward, Appl. Environ. Microbiol.57:419-25 (1991)). Based on process economics, this temperature shiftshould likely occur after the first phase of the two-stage fermentation,when the bulk of the organisms' growth has occurred.

It is contemplated that a variety of fermentation process designs may beapplied, where commercial production of omega fatty acids using theinstant Δ15 desaturase genes is desired. For example, commercialproduction of PUFAs from a recombinant microbial host may be produced bya batch, fed-batch or continuous fermentation process.

A batch fermentation process is a closed system wherein the mediacomposition is set at the beginning of the process and not subject tofurther additions beyond those required for maintenance of pH and oxygenlevel during the process. Thus, at the beginning of the culturingprocess the media is inoculated with the desired organism and growth ormetabolic activity is permitted to occur without adding additionalsubstrates (i.e., carbon and nitrogen sources) to the medium. In batchprocesses the metabolite and biomass compositions of the system changeconstantly up to the time the culture is terminated. In a typical batchprocess, cells moderate through a static lag phase to a high-growth logphase and finally to a stationary phase, wherein the growth rate isdiminished or halted. Left untreated, cells in the stationary phase willeventually die. A variation of the standard batch process is thefed-batch process, wherein the substrate is continually added to thefermentor over the course of the fermentation process. A fed-batchprocess is also suitable in the present invention. Fed-Batch processesare useful when catabolite repression is apt to inhibit the metabolismof the cells or where it is desirable to have limited amounts ofsubstrate in the media at any one time. Measurement of the substrateconcentration in fed-batch systems is difficult and therefore may beestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases (e.g., CO₂).Batch and fed-batch culturing methods are common and well known in theart and examples may be found in Thomas D. Brock in Biotechnoloyy: ATextbook of Industrial Microbiology, 2^(nd) ed., (1989) SinauerAssociates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992), herein incorporated by reference.

Commercial production of omega fatty acids using the instant Δ15desaturases may also be accomplished by a continuous fermentationprocess wherein a defined media is continuously added to a bioreactorwhile an equal amount of culture volume is removed simultaneously forproduct recovery. Continuous cultures generally maintain the cells inthe log phase of growth at a constant cell density. Continuous orsemi-continuous culture methods permit the modulation of one factor orany number of factors that affect cell growth or end productconcentration. For example, one approach may limit the carbon source andallow all other parameters to moderate metabolism. In other systems, anumber of factors affecting growth may be altered continuously while thecell concentration, measured by media turbidity, is kept constant.Continuous systems strive to maintain steady state growth and thus thecell growth rate must be balanced against cell loss due to media beingdrawn off the culture. Methods of modulating nutrients and growthfactors for continuous culture processes, as well as techniques formaximizing the rate of product formation, are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

Purification of PUFAs

The PUFAs may be found in the host microorganism as free fatty acids orin esterified forms such as acylglycerols, phospholipids, sulfolipids orglycolipids, and may be extracted from the host cell through a varietyof means well-known in the art. One review of extraction techniques,quality analysis and acceptability standards for yeast lipids is that ofZ. Jacobs (Critical Reviews in Biotechnology 12(5/6):463491 (1992)). Abrief review of downstream processing is also available by A. Singh andO. Ward (Adv. Appl. Microbiol. 45:271-312 (1997)).

In general, means for the purification of PUFAs may include extractionwith organic solvents, sonication, supercritical fluid extraction (e.g.,using carbon dioxide), saponification, and physical means such aspresses, or combinations thereof. Of particular interest is extractionwith methanol and chloroform in the presence of water (E. G. Bligh & W.J. Dyer, Can. J. Biochem. Physiol. 37:911-917 (1959)). Where desirable,the aqueous layer can be acidified to protonate negatively-chargedmoieties and thereby increase partitioning of desired products into theorganic layer. After extraction, the organic solvents can be removed byevaporation under a stream of nitrogen. When isolated in conjugatedforms, the products may be enzymatically or chemically cleaved torelease the free fatty acid or a less complex conjugate of interest, andcan then be subject to further manipulations to produce a desired endproduct. Desirably, conjugated forms of fatty acids are cleaved withpotassium hydroxide.

If further purification is necessary, standard methods can be employed.Such methods may include extraction, treatment with urea, fractionalcrystallization, HPLC, fractional distillation, silica gelchromatography, high-speed centrifugation or distillation, orcombinations of these techniques. Protection of reactive groups, such asthe acid or alkenyl groups, may be done at any step through knowntechniques (e.g., alkylation or iodination). Methods used includemethylation of the fatty acids to produce methyl esters. Similarly,protecting groups may be removed at any step. Desirably, purification offractions containing GLA, STA, ARA, DHA and EPA may be accomplished bytreatment with urea and/or fractional distillation.

Production pf ω-3 and/or ω-6 Fatty Acids in Plants

The coding regions of the invention can be expressed in plants, inparticular, oilseed plants. This is accomplished by: 1.) construction ofchimeric genes (comprising a Δ15 desaturase of the present inventionunder the control of suitable regulatory sequences such as promoters and3′ transcription terminators); 2.) transformation of the chimeric genesinto appropriate plant hosts; and 3.) expression of said chimeric genesfor production of PUFAs.

Thus, the instant invention concerns a recombinant construct foraltering the total fatty acid profile of mature seeds of an oilseedplant to produce an oil having an omega 3:omega 6 ratio of greater than0.4, said construct comprising an isolated nucleic acid fragmentselected from the group consisting of:

-   -   (a) an isolated nucleic acid fragment encoding all or part of        the amino acid sequence as set forth in SEQ ID NO:2;    -   (b) an isolated nucleic acid fragment that hybridizes with (a)        when washed with 0.1×SSC, 0.1% SDS, 65° C.;    -   (c) an isolated nucleic acid fragment encoding an amino acid        sequence having at least 46.2% sequence identity with the amino        acid sequences set forth in SEQ ID NOs:2, 6, 10, 14,18 based on        the Clustal V method of alignment; or    -   (d) an isolated nucleic acid fragment that is completely        complementary to (a), (b), or (c)        wherein said isolated nucleic acid fragment is operably linked        to at least one regulatory sequence.

The ratio of omega3 to omega6 can range from about 2:5 to at least about45:1. Useful ratios include but are not limited to omega3 from about 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 23, 24, 27,29, 31, 36, 42 and 45, versus omega 6 of about one. Other useful omega3to omega 6 ratios include, but are not limited to 2:5, 3:5, 4:5, 1:1,and 2:1. It is believed that any integer ratio of omega 3 to omega6 fromat least about 2:5 to at least about 45:1 would be useful.

The isolated nucleic acid fragment described herein that was isolatedfrom Fusarium moniliforme can be used to practice the invention.

This invention also concerns oilseed plants, plant cells, plant tissuesand/or plant parts comprising in their genome the recombinant constructof the invention.

In still a further aspect, this invention also concerns seeds obtainedfrom these transformed oilseed plants, oil obtained from these seeds,products obtained from the processing of the oil, use of this oil infood, animal feed or an industrial application, use of the by-productsin food or animal feed.

The present invention provides a variety of plant hosts fortransformation with the Δ-15 desaturases described herein. Plants sotransformed can be monocotyledonous plants or dicotyledonous plants, andpreferably they belong to a class of plants identified as oleaginous(e.g., oilseed plants). Examples of preferred oilseed plant hostsinclude, but are not limited to, soybean (Glycine and Soja sp.), corn(Zea mays), flax (Linum sp.), rapeseed (Brassica sp.), primrose, canola,maize, safflower (Carthamus sp.) and sunflower (Helianthus sp.).

Genetically, modified plants of the present invention are produced byoverexpression of the instant Δ-15 desaturases. This may be accomplishedby first constructing chimeric genes in which the Δ15 desaturase codingregion is operably-linked to control sequences capable of directingexpression of the gene in the desired tissues at the desired stage ofdevelopment. These control sequences may comprise a promoter, enhancer,silencer, intron sequences, 3′UTR and/or 5′UTR regions, and proteinand/or RNA stabilizing elements. Such elements may vary in theirstrength and specificity. For reasons of convenience, the chimeric genesmay comprise promoter sequences and translation leader sequences derivedfrom the same genes. 3′ Non-coding sequences encoding transcriptiontermination signals must also be provided. It is preferred that thechimeric gene be introduced via a vector and that the vector harboringthe Δ15 desaturase sequence also contain one or more selectable markergenes so that cells transformed with the chimeric gene can be selectedfrom non-transformed cells.

The present invention makes use of a variety of plant promoters to drivethe expression of the Δ15 desaturase gene(s) described herein orfunctional fragments thereof. Any promoter functional in a plant will besuitable, including (but not limited to): constitutive plant promoters,plant tissue-specific promoters, plant development-stage specificpromoters, inducible plant promoters, viral promoters, malegermline-specific promoters, female germline-specific promoters,flower-specific promoters and vegetative shoot apical meristem-specificpromoters.

As was noted above, a promoter is a DNA sequence that directs cellularmachinery of a plant to produce RNA from the contiguous coding sequencedownstream (3′) of the promoter. The promoter region influences therate, developmental stage, and cell type in which the RNA transcript ofthe gene is made. The RNA transcript is processed to produce messengerRNA (mRNA) which serves as a template for translation of the RNAsequence into the amino acid sequence of the encoded polypeptide. The 5′non-translated leader sequence is a region of the mRNA upstream of theprotein coding region that may play a role in initiation and translationof the mRNA. The 3′ transcription termination/polyadenylation signal isa non-translated region downstream of the protein coding region thatfunctions in the plant cells to cause termination of the RNA transcriptand the addition of polyadenylate nucleotides to the 3′ end of the RNA.

The origin of the promoter chosen to drive expression of the codingsequence is not important as long as it has sufficient transcriptionalactivity to accomplish the invention by expressing translatable mRNA forthe desired nucleic acid fragments in the desired host tissue at theright time. Either heterologous or non-heterologous (i.e., endogenous)promoters can be used to practice the invention.

Suitable promoters which can be used to practice the invention include,but are not limited to, the alpha prime subunit of beta conglycininpromoter, Kunitz trypsin inhibitor 3 promoter, annexin promoter, Gly1promoter, beta subunit of beta conglycinin promoter, P34/Gly Bd m 30Kpromoter, albumin promoter, Leg A1 promoter and Leg A2 promoter. Thelevel of activity of the annexin, or P34, promoter is comparable to thatof many known strong promoters, such as the CaMV 35S promoter(Atanassova et al., (1998) Plant Mol. Biol. 37:275-285; Battraw andHall, (1990) Plant Mol. Biol. 15:527-538; Holtorf et al., (1995) PlantMol. Biol. 29:637-646; Jefferson et al., (1987) EMBO J. 6:3901-3907;Wilmink et al., (1995) Plant Mol Biol. 28:949-955), the Arabidopsisoleosin promoters (Plant et al., (1994) Plant Mol. Biol. 25:193-205; Li,(1997) Texas A&M University Ph.D. dissertation, pp.107-128), theArabidopsis ubiquitin extension protein promoters (Callis et al., 1990),a tomato ubiquitin gene promoter (Rollfinke et al., 1998), a soybeanheat shock protein promoter (Schoffl et al., 1989), and a maize H3histone gene promoter (Atanassova et al., 1998).

Expression of chimeric genes in most plant cells makes the annexin orP34 promoter, which constitutes the subject matter of WO 2004/071178,published on Aug. 26, 2004 especially useful when seed specificexpression of a target heterologous nucleic acid fragment is required.Another useful feature of the annexin promoter is its expression profilein developing seeds. The annexin promoter of the invention is mostactive in developing seeds at early stages (before 10 days afterpollination) and is largely quiescent in later stages. The expressionprofile of the annexin promoter is different from that of manyseed-specific promoters, e.g., seed storage protein promoters, whichoften provide highest activity in later stages of development (Chen etal., (1989) Dev. Genet. 10:112-122; Ellerstrom et al., (1996) Plant Mol.Biol. 32:1019-1027; Keddie et al., (1994) Plant Mol. Biol. 24:327-340;Plant et al., (1994) Plant Mol. Biol. 25:193-205; Li, (1997) Texas A&MUniversity Ph.D. dissertation, pp.107-128). The P34 promoter has a moreconventional expression profile but remains distinct from other knownseed specific promoters. Thus, the annexin, or P34, promoter will be avery attractive candidate when overexpression, or suppression, of a genein embryos is desired at an early developing stage. For example, it maybe desirable to overexpress a gene regulating early embryo developmentor a gene involved in the metabolism prior to seed maturation.

The promoter is then operably linked in a sense orientation usingconventional means well known to those skilled in the art.

Once the recombinant construct has been made, it may then be introducedinto the oilseed plant cell of choice by methods well known to those ofordinary skill in the art including, for example, transfection,transformation and electroporation as described above. The transformedplant cell is then cultured and regenerated under suitable conditionspermitting expression of the PUFA which is then recovered and purified.

The recombinant constructs of the invention may be introduced into oneplant cell or, alternatively, each construct may be introduced intoseparate plant cells.

Expression in a plant cell may be accomplished in a transient or stablefashion as is described above.

The desired PUFAs can be expressed in seed. Also within the scope ofthis invention are seeds or plant parts obtained from such transformedplants.

Plant parts include differentiated and undifferentiated tissues,including but not limited to, roots, stems, shoots, leaves, pollen,seeds, tumor tissue, and various forms of cells and culture such assingle cells, protoplasts, embryos, and callus tissue. The plant tissuemay be in plant or in organ, tissue or cell culture.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published, amongothers, for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135);soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al. (1996) Plant Cell Rep.15:653-657, McKently et al. (1995) Plant Cell Rep. 14:699-703); papaya(Ling, K. et al. (1991) Bio/technology 9:752-758); and pea (Grant et al.(1995) Plant Cell Rep. 15:254-258). For a review of other commonly usedmethods of plant transformation see Newell, C. A. (2000) Mol.Biotechnol. 16:53-65. One of these methods of transformation usesAgrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F. (1987)Microbiol. Sci. 4:24-28). Transformation of soybeans using directdelivery of DNA has been published using PEG fusion (PCT publication WO92/17598), electroporation (Chowrira, G. M. et al. (1995) Mol.Biotechnol. 3:17-23; Christou, P. et al. (1987) Proc. Natl. Acad. Sci.U.S.A. 84:3962-3966), microinjection, or particle bombardment (McCabe,D. E. et. al. (1988) Bio/Technology 6:923; Christou et al. (1988) PlantPhysiol. 87:671-674).

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, (1988) In.:Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., SanDiego, Calif.). This regeneration and growth process typically includesthe steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant DNA fragments and recombinant expressionconstructs and the screening and isolating of clones, (see for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press; Maliga et al. (1995) Methods in Plant MolecularBiology, Cold Spring Harbor Press; Birren et al. (1998) Genome Analysis:Detecting Genes, 1, Cold Spring Harbor, New York; Birren et al. (1998)Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, New York; PlantMolecular Biology: A Laboratory Manual, eds. Clark, Springer, N.Y.(1997)).

In another aspect, this invention concerns a method for increasing theratio of omega-3 fatty acids to omega-6 fatty acids in an oilseed plantcomprising:

-   -   a) transforming an oilseed plant cell of with the recombinant        construct of the invention    -   b) regenerating an oilseed plant from the transformed plant cell        of step (a);    -   c) selecting those transformed plants having an increased ratio        of omega-3 fatty acids to omega-6 fatty acid compared to the        ratio of omega-3 fatty acids to omega-6 fatty acid in an        untransformed plant.

In still a further aspect, this invention concerns a method forproducing alpha-linolenic acid in seed of an oilseed plant wherein thealpha-linolenic acid content of the oil in the seed is at least 25% ofthe total fatty acid content of the seed oil, said method comprising:

-   -   a) transforming an oilseed plant cell of with the recombinant        construct of the invention    -   b) regenerating an oilseed plant from the transformed plant cell        of step (a);    -   c) selecting those transformed plants having at least 25%        alpha-linolenic acid of the total fatty acid content of the seed        oil.

The alpha-linolenic content of the oil in such seeds can range from atleast 25% to about 89% or any integer percentage between 25% and 89%,e.g., 26%, 27%, etc.

The invention also concerns oilseed plants, plant cells, plant tissuesand/or plant parts comprising in their genome the recombinant constructof the invention made by the methods of this invention.

In still a further aspect, this invention also concerns seeds obtainedfrom these transformed oilseed plants, oil obtained from these seeds,products obtained from the processing of the oil, use of this oil infood, animal feed or an industrial application, use of the by-productsin food or animal feed.

Methods of isolating seed oils are well known in the art: (Young et al,Processing of Fats and Oils, in “The Lipid Handbook” (Gunstone et aleds.) Chapter 5 pp 253-257; London, Chapman & Hall, 1994).

The altered seed oils can then be added to nutritional compositions suchas a nutritional supplement, food products, infant formula, animal feed,pet food and the like.

Compared to other vegetable oils, the oils of the invention are believedto function similarly to other oils in food applications from a physicalstandpoint. Partially hydrogenated oils, such as soybean oil, are widelyused as ingredients for soft spreads, margarine and shortenings forbaking and frying.

Examples of food products or food analogs into which altered seed oilsor altered seeds of the invention may be incorporated include a meatproduct such as a processed meat product, a cereal food product, a snackfood product, a baked goods product, a fried food product, a health foodproduct, an infant formula, a beverage, a nutritional supplement, adairy product, a pet food product, animal feed or an aquaculture foodproduct. Food analogs can be made use processes well known to thoseskilled in the art. U.S. Pat. Nos. 6,355,296 B1 and 6,187,367 B1describe emulsified meat analogs and emulsified meat extenders. U.S.Pat. No. 5,206,050 B1 describes soy protein curd useful for cooked foodanalogs (also can be used as a process to form a curd useful to makefood analogs). U.S. Pat. No. 4,284,656 to Hwa describes a soy proteincurd useful for food analogs. U.S. Pat. No. 3,988,485 to Hibbert et al.describes a meat-like protein food formed from spun vegetable proteinfibers. U.S. Pat. No. 3,950,564 to Puski et al. describes a process ofmaking a soy based meat substitute and U.S. Pat. No. 3,925,566 toReinhart et al. describes a simulated meat product. For example, soyprotein that has been processed to impart a structure, chunk or fiberfor use as a food ingredient is called “textured soy protein” (TSP).TSPs are frequently made to resemble meat, seafood, or poultry instructure and appearance when hydrated.

There can be mentioned meat analogs, cheese analogs, milk analogs andthe like.

Meat analogs made from soybeans contain soy protein or tofu and otheringredients mixed together to simulate various kinds of meats. Thesemeat alternatives are sold as frozen, canned or dried foods. Usually,they can be used the same way as the foods they replace. Meatalternatives made from soybeans are excellent sources of protein, ironand B vitamins. Examples of meat analogs include, but are not limitedto, ham analogs, sausage analogs, bacon analogs, and the like.

Food analogs can be classified as imitiation or substitutes depending ontheir functional and compositional characteristics. For example, animitation cheese need only resemble the cheese it is designed toreplace. However, a product can generally be called a substitute cheeseonly if it is nutritionally equivalent to the cheese it is replacing andmeets the minimum compositional requirements for that cheese. Thus,substitute cheese will often have higher protein levels than imitationcheeses and be fortified with vitamins and minerals.

Milk analogs or nondairy food products include, but are not limited to,imitation milk, nondairy frozen desserts such as those made fromsoybeans and/or soy protein products.

Meat products encompass a broad variety of products. In the UnitedStates “meat” includes “red meats” produced from cattle, hogs and sheep.In addition to the red meats there are poultry items which includechickens, turkeys, geese, guineas, ducks and the fish and shellfish.There is a wide assortment of seasoned and processes meat products:fresh, cured and fried, and cured and cooked. Sausages and hot dogs areexamples of processed meat products. Thus, the term “meat products” asused herein includes, but is not limited to, processed meat products.

A cereal food product is a food product derived from the processing of acereal grain. A cereal grain includes any plant from the grass familythat yields an edible grain (seed). The most popular grains are barley,corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat andwild rice. Examples of a cereal food product include, but are notlimited to, whole grain, crushed grain, grits, flour, bran, germ,breakfast cereals, extruded foods, pastas, and the like.

A baked goods product comprises any of the cereal food productsmentioned above and has been baked or processed in a manner comparableto baking, i.e., to dry or harden by subjecting to heat. Examples of abaked good product include, but are not limited to bread, cakes,doughnuts, bread crumbs, baked snacks, mini-biscuits, mini-crackers,mini-cookies, and mini-pretzels. As was mentioned above, oils of theinvention can be used as an ingredient.

In general, soybean oil is produced using a series of steps involvingthe extraction and purification of an edible oil product from the oilbearing seed. Soybean oils and soybean byproducts are produced using thegeneralized steps shown in the diagram below.

Soybean seeds are cleaned, tempered, dehulled, and flaked whichincreases the efficiency of oil extraction. Oil extraction is usually

accomplished by solvent (hexane) extraction but can also be achieved bya combination of physical pressure and/or solvent extraction. Theresulting oil is called crude oil. The crude oil may be degummed byhydrating phospholipids and other polar and neutral lipid complexes thatfacilitate their separation from the nonhydrating, triglyceride fraction(soybean oil). The resulting lecithin gums may be further processed tomake commercially important lecithin products used in a variety of foodand industrial products as emulsification and release (antisticking)agents. The term lecithin itself has different meanings when used inchemistry and biochemistry than when used commercially. Chemically,lecithin is phosphatidylcholine. Commercially, it refers to a naturalmixture of neutral and polar lipids. Phosphatidylcholine, which is apolar lipid, is present in commercial lecithin in concentrations of 20to 90%. Lecithins containing phosphatidylcholine are produced fromvegetable, animal and microbial sources, but mainly from vegetablesources. Soybean, sunflower and rapeseed are the major plant sources ofcommercial lecithin. Soybean is the most common source. Plant lecithinsare considered to be GRAS (generally regarded as safe). Degummed oil maybe further refined for the removal of impurities; primarily free fattyacids, pigments, and residual gums. Refining is accomplished by theaddition of a caustic agent that reacts with free fatty acid to formsoap and hydrates phosphatides and proteins in the crude oil. Water isused to wash out traces of soap formed during refining. The soapstockbyproduct may be used directly in animal feeds or acidulated to recoverthe free fatty acids. Color is removed through adsorption with ableaching earth that removes most of the chlorophyll and carotenoidcompounds. The refined oil can be hydrogenated resulting in fats withvarious melting properties and textures. Winterization (fractionation)may be used to remove stearine from the hydrogenated oil throughcrystallization under carefully controlled cooling conditions.Deodorization which is principally steam distillation under vacuum, isthe last step and is designed to remove compounds which impart odor orflavor to the oil. Other valuable byproducts such as tocopherols andsterols may be removed during the deodorization process. Deodorizeddistillate containing these byproducts may be sold for production ofnatural vitamin E and other high-value pharmaceutical products. Refined,bleached, (hydrogenated, fractionated) and deodorized oils and fats maybe packaged and sold directly or further processed into more specializedproducts. A more detailed reference to soybean seed processing, soybeanoil production and byproduct utilization can be found in Erickson, 1995,Practical Handbook of Soybean Processing and Utilization, The AmericanOil Chemists' Society and United Soybean Board.

Soybean oil is liquid at room temperature because it is relatively lowin saturated fatty acids when compared with oils such as coconut, palm,palm kernel and cocoa butter. Many processed fats, including spreads,confectionary fats, hard butters, margarines, baking shortenings, etc.,require varying degrees of solidity at room temperature and can only beproduced from soybean oil through alteration of its physical properties.This is most commonly achieved through catalytic hydrogenation.

Hydrogenation is a chemical reaction in which hydrogen is added to theunsaturated fatty acid double bonds with the aid of a catalyst such asnickel. High oleic soybean oil contains unsaturated oleic, linoleic, andlinolenic fatty acids and each of these can be hydrogenated.Hydrogenation has two primary effects. First, the oxidative stability ofthe oil is increased as a result of the reduction of the unsaturatedfatty acid content. Second, the physical properties of the oil arechanged because the fatty acid modifications increase the melting pointresulting in a semi-liquid or solid fat at room temperature.

There are many variables which affect the hydrogenation reaction whichin turn alter the composition of the final product. Operating conditionsincluding pressure, temperature, catalyst type and concentration,agitation and reactor design are among the more important parameterswhich can be controlled. Selective hydrogenation conditions can be usedto hydrogenate the more unsaturated fatty acids in preference to theless unsaturated ones. Very light or brush hydrogenation is oftenemployed to increase stability of liquid oils. Further hydrogenationconverts a liquid oil to a physically solid fat. The degree ofhydrogenation depends on the desired performance and meltingcharacteristics designed for the particular end product. Liquidshortenings, used in the manufacture of baking products, solid fats andshortenings used for commercial frying and roasting operations, and basestocks for margarine manufacture are among the myriad of possible oiland fat products achieved through hydrogenation. A more detaileddescription of hydrogenation and hydrogenated products can be found inPatterson, H. B. W., 1994, Hydrogenation of Fats and Oils: Theory andPractice. The American Oil Chemists' Society.

Hydrogenated oils have also become controversial due to the presence oftrans fatty acid isomers that result from the hydrogenation process.Ingestion of large amounts of trans isomers has been linked withdetrimental health effects including increased ratios of low density tohigh density lipoproteins in the blood plasma and increased risk ofcoronary heart disease.

A snack food product comprises any of the above or below described foodproducts.

A fried food product comprises any of the above or below described foodproducts that has been fried.

A health food product is any food product that imparts a health benefit.Many oilseed-derived food products may be considered as health foods.

The beverage can be in a liquid or in a dry powdered form.

For example, there can be mentioned non-carbonated drinks; fruit juices,fresh, frozen, canned or concentrate; flavored or plain milk drinks,etc. Adult and infant nutritional formulas are well known in the art andcommercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum®from Ross Products Division, Abbott Laboratories).

Infant formulas are liquids or reconstituted powders fed to infants andyoung children. They serve as substitutes for human milk. Infantformulas have a special role to play in the diets of infants becausethey are often the only source of nutrients for infants. Althoughbreast-feeding is still the best nourishment for infants, infant formulais a close enough second that babies not only survive but thrive. Infantformula is becoming more and more increasingly close to breast milk.

A dairy product is a product derived from milk. A milk analog ornondairy product is derived from a source other than milk, for example,soymilk as was discussed above. These products include, but are notlimited to, whole milk, skim milk, fermented milk products such asyogurt or sour milk, cream, butter, condensed milk, dehydrated milk,coffee whitener, coffee creamer, ice cream, cheese, etc.

A pet food product is a product intended to be fed to a pet such as adog, cat, bird, reptile, fish, rodent and the like. These products caninclude the cereal and health food products above, as well as meat andmeat byproducts, soy protein products, grass and hay products, includingbut not limited to alfalfa, timothy, oat or brome grass, vegetables andthe like.

Animal feed is a product intended to be fed to animals such as turkeys,chickens, cattle and swine and the like. As with the pet foods above,these products can include cereal and health food products, soy proteinproducts, meat and meat byproducts, and grass and hay products as listedabove.

Aqualculture feed is a product intended to be used in aquafarming whichconcerns the propagation, cultivation or farming of aquatic organisms,animals and/or plants in fresh or marine waters.

In yet another embodiment, this invention includes oil obtained from theseeds of such plants.

In yet another aspect, the invention concerns a recombinant constructfor altering the total fatty acid profile of mature seeds of an oilseedplant to produce an oil having an omega 3 to omega 6 ratio greater than2, wherein said oil has an eicosapentaenoic acid content greater than2%, said construct comprising an isolated nucleic acid fragment selectedfrom the group consisting of:

-   -   (a) an isolated nucleic acid fragment encoding all or part of        the amino acid sequence as set forth in SEQ ID NO:2;    -   (b) an isolated nucleic acid fragment that hybridizes with (a)        when washed with 0.1×SSC, 0.1% SDS, 65° C.;    -   (c) an isolated nucleic acid fragment encoding an amino acid        sequence having at least 46.2% sequence identity with the amino        acid sequences set forth in SEQ ID NOs:2, 6, 10, 14,18 based on        the Clustal V method of alignment; or    -   (d) an isolated nucleic acid fragment that is completely        complementary to (a), (b), or (c)        wherein said isolated nucleic acid fragment is operably linked        to at least one regulatory sequence.

Also, this invention concerns oilseed plants, plant cells, planttissues, or plant parts comprising in their genomes the recombinantconstruct of the invention. The invention also concerns the seedsobtained from such plants, oil obtained from these seeds, use of thisoil in food or animal feed, by-products obtained from the processing ofthis oil and use of these by-products in food or animal feed.

Additionally the invention provides microbial oils produced by themethods of the invention.

In still another aspect, the present invention concerns a method forproducing eicosapentaenoic acid in seed of an oilseed plant to producean oil having an omega 3 to omega 6 ratio greater than 2, wherein saidoil has an eicosapentaenoic acid content greater than 2% of the totalfatty acid content of the seed oil, said method comprising:

-   -   a) transforming an oilseed plant cell of with the recombinant        construct of the present invention;    -   b) regenerating an oilseed plant from the transformed plant cell        of step (a);    -   c) selecting those transformed plants having at least 2%        eicosapentaenoic acid of the total fatty acid content of the        seed oil.

Additionally, this invention concerns oilseed plants, plant cells, planttissues, or plant parts comprising in their genomes the recombinantconstruct of the invention. The invention also concerns the seedsobtained from such plants, oil obtained from these seeds, use of thisoil in food or animal feed, by-products obtained from the processing ofthis oil and use of these by-products in food or animal feed.

Additionally the invention provides microbial oils produced by themethods of the invention.

Various plasmids and vectors comprising the chimeric Al5 desaturasegenes can then be constructed, using methods which are well known tothose skilled in the art; see, for example, the techniques described inSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989(hereinafter “Maniatus”); and by Ausubel et al., Current Protocols inMolecular Biology, published by Greene Publishing Assoc. andWiley-Interscience (1987).

The choice of a plasmid vector depends upon the method that will be usedto transform host plants. The skilled artisan is well aware of thegenetic elements that must be present on the plasmid vector in order tosuccessfully transform, select and propagate host cells containing thechimeric gene. For example, the termination signals usually employed arefrom the Nopaline Synthase promoter or from the CAMV 35S promoter. Aplant translational enhancer often used is the tobacco mosaic virus(TMV) omega sequences; additionally, the inclusion of an intron (e.g.,Intron-1 from the Shrunken gene of maize) has been shown to increaseexpression levels by up to 100-fold (Mait, Transgenic Res. 6:143-156(1997); Ni, Plant Journal 7:661-676 (1995)). Additional regulatoryelements may include transcriptional (as well as translational)enhancers.

In addition to the regulatory elements described above for a preferredexpression vector, it is also useful for the vector to comprise aselectable and/or scorable marker. Preferably, the marker gene is anantibiotic resistance gene whereby the appropriate antibiotic can beused to select for transformed cells from among those cells that werenot transformed. Selectable marker genes useful for the selection oftransformed plant cells, callus, plant tissue and plants are well knownto those skilled in the art. Examples include, but are not limited to:npt, which confers resistance to the aminoglycosides neomycin, kanamycinand paromycin; hygro, which confers resistance to hygromycin; trpB,which allows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman, Proc.Natl. Acad. Sci. USA 85:8047 (1988)); mannose-6-phosphate isomerase,which allows cells to utilize mannose (WO 94/20627); ODC (ornithinedecarboxylase), which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine (or “DFMO”; McConlogue, In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y. (1987)); and deaminase fromAspergillus terreus, which confers resistance to blasticidin S (Tamura,Biosci. Biotechnol. Biochem. 59 2336-2338 (1995)).

Useful scorable markers are also known to those skilled in the art andare commercially available, such as the genes encoding luciferase(Giacomin, P I. Sci. 116:59-72 (1996); Scikantha, J. Bact. 178:121(1996)), green fluorescent protein (Gerdes, FEBS Lett. 389:44-47 (1996))or R-glucuronidase (Jefferson, EMBO J. 6:3901-3907 (1987)). Thisembodiment is particularly useful for simple and rapid screening ofcells, tissues and organisms containing a vector comprising a Δ15desaturase.

For some applications it may be useful to direct the Δ15 desaturaseproteins to different cellular compartments. It is thus envisioned thatthe chimeric genes described above may be further modified by theaddition of appropriate intracellular targeting sequences to theircoding regions (and/or with targeting sequences that are already presentremoved). These additional targeting sequences include chloroplasttransit peptides (Keegstra et al., Cell 56:247-253 (1989)), signalsequences that direct proteins to the endoplasmic reticulum (Chrispeelset al., Ann. Rev. Plant Phys. Plant Mol. 42:21-53 (1991)), and nuclearlocalization signals (Raikhel et al., Plant Phys. 100:1627-1632 (1992)).While the references cited give examples of each of these, the list isnot exhaustive and more targeting signals of utility may be discoveredin the future which are useful in the invention.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques include transformation with DNA employing Agrobacteriumtumefaciens or A. rhizogenes as the transforming agent. It isparticularly preferred to use the binary type vectors of Ti and Riplasmids of Agrobacterium spp. Ti-derived vectors transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants such as soybean, cotton, rape, tobacco and rice (Pacciotti etal., Bio/Technology 3:241 (1985); Byrne et al., Plant Cell, Tissue andOrgan Culture 8:3 (1987); Sukhapinda et al., Plant Mol. Biol. 8:209-216(1987); Lorz et al., Mol. Gen. Genet. 199:178 (1985); Potrykus, Mol.Gen. Genet. 199:183 (1985); Park et al., J. Plant Biol. 38(4):365-71(1995); Hiei et al., Plant J. 6:271-282 (1994)). The use of T-DNA totransform plant cells has received extensive study and is amplydescribed (EP 120516; Hoekema, In: The Binary Plant Vector System,Offset-drukkerij Kanters B. V.; Alblasserdam (1985), Chapter V; Knauf etal., Genetic Analysis of Host Range Expression by Agrobacterium, In:Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. Ed.;Springer-Verlag: N.Y., 1983, p 245; and An et al., EMBO J. 4:277-284(1985)). For introduction into plants, the chimeric genes of theinvention can be inserted into binary vectors as described in theExamples.

Other transformation methods are available to those skilled in the art,such as: 1.) direct uptake of foreign DNA constructs (see EP 295959);2.) techniques of electroporation (see Fromm et al., Nature (London)319:791 (1986)); 3.) high-velocity ballistic bombardment with metalparticles coated with the nucleic acid constructs (see Kline et al.,Nature (London) 327:70 (1987) and U.S. Pat. No.4,945,050); or 4.)microinjection (see Gene Transfer To Plants, Potrykus and Spangenberg,Eds., Springer Verlag: Berlin, N.Y. (1995)). For a review of commonlyused methods of plant transformation see Newell, C. A. (Mol. Biotechnol.16:53-65 (2000)). The transformation of most dicotyledonous plants ispossible with the methods described above; however, additionaltransformation techniques have been developed for the successfultransformation of monocotyledonous plants. These include protoplasttransformation and transformation by an in planta method usingAgrobacterium tumefaciens. This in planta method (Bechtold andPelletier, C. R. Acad. Sci. Paris, 316:1194 (1993); or Clough S. J.,Bent A. F.; Plant Journal 16(6):735-43 (1998)) involves the applicationof A. tumefaciens to the outside of the developing flower bud and thenintroduction of the binary vector DNA to the developing microsporeand/or macrospore and/or developing seed, so as to produce a transformedseed without the exogenous application of cytokinin and/or gibberellin.Those skilled in the art will be aware that the selection of tissue foruse in such a procedure may vary; however, it is preferable generally touse plant material at the zygote formation stage for in plantatransformation procedures.

Once transformed, there are a variety of methods for the regeneration ofplants from plant tissue. The particular method of regeneration willdepend on the starting plant tissue and the particular plant species tobe regenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Methods for Plant Molecular Biology;Weissbach and Weissbach, Eds., Academic: San Diego, Calif. (1988)). Ofparticular relevance are methods to transform foreign genes intocommercially important oilseed crops, such as rapeseed (see De Block etal., Plant Physiol. 91:694-701 (1989); U.S. Pat. No. 5,463,174),sunflower (Everett et al., Bio/Technology 5:1201 (1987)), soybean(McCabe et al., Bio/Technology 6:923 (1988); Hinchee et al.,Bio/Technology 6:915 (1988); Chee et al., Plant Physiol. 91:1212-1218(1989); Christou et al., Proc. Natl. Acad. Sci USA 86:7500-7504 (1989);EP 301749; U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011) and corn(Gordon-Kamm et al., Plant Cell 2:603-618 (1990); Fromm et al.,Biotechnology 8:833-839 (1990)).

Typically, transgenic plant cells are placed in an appropriate selectivemedium for selection of transgenic cells that are then grown to callus.Shoots are grown from callus and plantlets generated from the shoot bygrowing in rooting medium. The various constructs normally will bejoined to a marker for selection in plant cells. Conveniently, themarker may be resistance to a biocide (particularly an antibiotic suchas kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide orthe like). The particular marker used will allow for selection oftransformed cells as compared to cells lacking the DNA that has beenintroduced.

One skilled in the art recognizes that the expression level andregulation of a transgene in a plant can vary significantly from line toline. Thus, one has to test several lines to find one with the desiredexpression level and regulation. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., EMBO J4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86(1989)), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA blots(Southern, J. Mol. Biol. 98: 503 (1975)), Northern analysis of mRNAexpression (Kroczek, J. Chromatogr. Biomed. Appl., 618(1-2):133-145(1993)), Western analysis of protein expression or phenotypic analysis.One particularly useful way to quantitate protein expression and todetect replication in different plant tissues is to use a reporter gene(e.g., GUS). Once transgenic plants have been obtained, they may begrown to produce plant tissues or parts having the desired phenotype.The plant tissue or plant parts may be harvested and/or the seedcollected. The seed may serve as a source for growing additional plantswith tissues or parts having the desired characteristics.

As was discussed above, methods of isolating seed oils are well known inthe art (Young et al., In The Lipid Handbook; Gunstone et al., Eds.;Chapman & Hall: London, 1994; pp 253-257). The altered seed oils canthen be used in various nutritional compositions (e.g., nutritionalsupplements, food products, infant formulas, animal feed, pet food,etc.).

The ultimate goal of the work described herein is the development of anorganism that accumulates oils enriched in ω-3 PUFAs, wherein onepreferred host is an oleaginous plant or an oleaginous yeast. Towardthis end, desaturases must be identified that function efficiently toenable synthesis and high accumulation of preferred ω-3 PUFAs in thesehosts. Identification of efficient Δ15 and ω-3 desaturases is alsonecessary for the manipulation of the ratio of ω-3 to ω-6 PUFAs producedin host cells.

In previous work, the native Yarrowia lipolytica Δ12 desaturase wasisolated and over-expressed this protein, resulting in increasedconversion of oleic acid to LA with respect to the wildtype cells (U.S.patent application Ser. No. 10/840325, incorporated entirely byreference; see also Example 2 herein and SEQ ID NOs:54 and 55). Despitethe increased availability of LA within these host cells, however, itwas desirable to obtain an even larger substrate pool suitable to enablehigh-level production of a variety of ω-3 PUFAs (e.g., EPA) within theY. lipolytica transformant cells. Thus, expression of a heterologousprotein having high-level Δ15 desaturase activity was thereforeadvantageous in the pathway engineering of the organism. Sincepreviously isolated Δ15 desaturases from plant sources were not expectedto function efficiently in oleaginous yeast, it was therefore an objectof the present invention to isolate a fungal Δ15 desaturase. It wasexpected that over-expression of this fungal desaturase would increasesubstrate pools of ALA within oleaginous yeast hosts, thereby permittingsynthesis and high accumulation of preferred ω-3 PUFAs (e.g., STA, ETA,EPA, DPA and DHA) in these hosts. Increased Δ15 desaturase activitywould also enable modification of the ratio of ω-3 to ω-6 PUFAs.

To achieve these goals, in the present invention Applicants isolated andcloned a DNA fragment from Fusarium moniliforme that encodes a Δ15desaturase enzyme (“Fm1”; SEQ ID NOs:1 and 2). Confirmation of thisgene's activity as a Δ15 desaturase was provided based upon theproduction of ALA in wild type Yarrowia lipolytica cells upontransformation with a chimeric gene comprising the F. moniliforme Fm1desaturase (Example 5, wherein the percent substrate conversioncalculated as [18:3]/[18:2+18:3]*100) was 82.5%).

Surprisingly, however, the F. moniliforme Δ15 desaturase also hasseveral unique characteristics, as compared to previously known Δ15desaturases. Specifically, in addition to the novel sequence of the F.moniliforme Δ5 desaturase, it is also distinguished by its significantΔ12 desaturase activity, % ALA product accumulation and broad substratespecificify.

Significant Δ12 Desaturase Activity

As shown in the Examples, the Fusarium moniliforme Δ15 desaturase (Fm1)disclosed herein has significant Δ12 desaturase activity (see Table 9,Example 5), wherein a Δ12 desaturase-disrupted strain of Yarrowialipolytica that was transformed with a chimeric gene encoding SEQ IDNO:2 was able to convert 24% of oleic acid to LA (percent substrateconversion calculated as ([18:2+18:3]/[18:1+18:2+18:3])*100), inaddition to 96% of LA to ALA (percent substrate conversion calculated as[18:3]/[18:2+18:3]*100)). This bifunctionality is in marked contrast toany other known Δ15 desaturase. And, although desaturases are known withspecificity toward more than one substrate, the bifunctionality of theF. moniliforme desaturase (wherein the protein possesses both Δ12 andΔ15 desaturase activity) distinguishes it from any known Δ12 or Δ15fatty acid desaturase identified to date.

Percent ALA Product Accumulation

The Fusarium moniliforme Δ15 desaturase disclosed herein enablesextremely high synthesis of ALA when expressed in Yarrowia lipolytica,relative to that described for other heterologously expressed Δ15desaturases (e.g., worms and plants). Specifically, the Fusarium enzymewas very active (i.e., Yarrowia lipolytica that was transformed with achimeric gene encoding SEQ ID NO:2 was able to demonstrate a % productaccumulation of ALA of 31%, relative to the total fatty acids in thetransformant host cell (see Table 9, Example 5)). This represents aconversion efficiency to ALA of 83% (calculated as[18:3]/[18:2+18:3]*100). In the Δ12 desaturase-disrupted strain ofYarrowia lipolytica that was transformed with a chimeric gene encodingSEQ ID NO:2, a conversion efficiency to ALA of 96% was demonstrated. Incontrast, the % product accumulation of ALA when expressing the C.elegans Δ15 desaturase in the non-oleaginous yeast Saccharomycescerevisiae was only 4.1% (Meesapyodsuk et al., Biochem. 39:11948-11954(2000)); and, the % product accumulation of ALA when expressing the B.napus Δ15 desaturase in S. cerevisiae was only 1.3% (Reed., D. W. etal., Plant Physiol. 122:715-720 (2000)).

The high efficiency of the Fusarium moniliforme Δ15 desaturase,especially in the Δ12 desaturase-disrupted strain of Y. lipolytica, isthe result of the protein's bifunctional Δ12 and Δ15 desaturaseactivity, whereby the product of the Δ12 desaturation is the substratefor the Δ15 desaturase. One skilled in the art would recognize that theratio of 18:3/18:2 could be maximized by expression of the enzymedisclosed herein in host organisms having little or no ability tosynthesize 18:2 (e.g., a Δ12 desaturase-null line in an oleaginous yeastor an Arabidopsis fad2 mutant).

Broad Substrate Specificity

Finally, the Fusarium moniliforme Δ15 enzyme has relatively broadsubstrate specificity on downstream ω-6 derivatives of 18:2;specifically, the Δ15 desaturase described herein is able to catalyzeconversion of GLA to STA, DGLA to ETA, and ARA to EPA. In contrast tothe heterologous expression of worm (C. elegans) and plant (B. napus)Δ15 desaturases in S. cerevisae (Meesapyodsuk et al., supra; Reed etal., supra), however, the Applicants' data herein demonstrate that theFusarium moniliforme Δ15 desaturase converts the ω-6 substrates to theirω-3 counterparts much more efficiently, i.e., with higher % substrateconversion, when expressed in Yarrowia (Table 4). TABLE 4 QualitativeComparison Of Substrate Preferences Of Δ15 Desaturases From Worm, PlantAnd Fungus Host Organism S. cerevisiae S. cerevisiae Y. lipolytica Δ15desaturase source C. elegans B. napus F. moniliforme ω-6 substrate %substrate conversion 18:2 (LA) 11.1 2.6 81.6 18:3 (GLA) 15.4 0.7 35.020:3 (DGLA) 5.9 0.6 20.0 20:4 (ARA) 1.9 0.7 ndNote:ω-6 substrate was fed in all cases except for 18:2 in Y. lipolytica;Nd = not determined

Thus, heterologous expression of the fungal Δ15 desaturase of theinvention increases cellular carbon flow into the ω-3 fatty acidbiosynthetic pathway, by enhancing the biosynthesis of ALA. As a result,the ratio of ω-3/ω-6 fatty acids is increased and production of moredownstream ω-3 fatty acids (e.g., STA, ETA and EPA) is enabled, whenother PUFA biosynthetic enzymes are co-expressed with the Δ15 desaturaseherein. It is expected that these results will occur in anymicroorganism in which the Δ15 desaturase of the present invention isexpressed. In alternative embodiments, the Applicants have demonstratedsimilar results by overexpression of the Fusarium moniliforme Δ15desaturase in plant oilseed hosts. Therefore, expression of the presentFusarium moniliforme Δ15 desaturase in any host cell is expected topermit the host cell to produce ALA at levels greater than about 10% ofthe total fatty acids, where greater than about 30% is preferable andgreater than about 50% is most preferred. Similarly, such transformantswill demonstrate altered ratios of ALA to LA where ratios of ALA:LA ofat least about 4 will be typical, ratios of at least 8 are preferred andratios of at least 12 are most preferred. Microbial oils extracted fromthese transformants will contain greater than about 10% ALA, wheregreater than about 30% is equally expected and greater than 50% isexpected to be typical.

Additionally, Applicants have also identified a suite of Al5 desaturasesorthologous to the Fusarium moniliforme protein described above fromAspergillus nidulans, Neurospora crassa, Magnaporthe grisea, andFusarium graminearium (i.e., SEQ ID NOs:6,14,10 and 18, respectively).These fungal proteins are also expected to be useful for expression Δ15desaturase activity in different host cells, including oleaginous yeast(e.g., Yarrowia lipolytica). These proteins (including the Fusariummoniliforme Δ12 desaturase (SEQ ID NO:2)) clustered within a distinctsub-family of proteins (referred to herein as “Sub-family 1”) that arewell-distinguished from the proteins clustered within “Sub-family 2”(i.e., SEQ ID NOs:4, 8, 12, 16 and 20, identified in co-pending U.S.Provisional Application 60/570679 as Δ12 desaturases), despite allproteins' identification as homologous to the Y. lipolytica Δ12desaturase identified herein as SEQ ID NO:55 (characterized inco-pending U.S. patent application Ser. No. 10/840325). Together, theproteins of sub-family 1 (identified herein as Δ15 desaturases)represent a group of proteins having at least 46.2% identity to oneanother (Example 3) and they are well-distinguished by sequence homologyfrom previously described Δ15 desaturases.

Functional characterization of the Aspergillus nidulans and Neurosporacrassa proteins, which confirmed their activity as Δ15 desaturases, isdescribed in WO 03/099216. Confirmation of the putative Magnaporthegrisea Δ15 desaturase (“Mg1”; SEQ ID NOs:9 and 10) gene's activity as aΔ15 desaturase was provided herein based upon the production of ALA inwild type Yarrowia lipolytica cells upon transformation with a chimericgene comprising Mg1 (Example 6). Comparison of the activity of these Δ15desaturases to that of the Fusarium moniliforme Δ15 desaturase describedabove, however, revealed that not all of the Δ15 desaturase proteins ofsub-family 1 were characterized as having bifunctional Δ12/Δ15desaturase activity. Specifically, based on the results provided in WO2003/099216, the Neurospora crassa and Aspergillus nidulans proteins didnot show bifunctional Δ12/Δ15 desaturase activity. In contrast, theMagnaporthe grisea protein behaved similarly to the Fusarium moniliformeprotein, and thus both were classified as having bifunctional Δ12/Δ15desaturase activity. It is hypothesized that the Fusarium graminearium(“Fg1”; SEQ ID NOs:17 and 18) will also have bifunctional Δ12/Δ15desaturase activity, since Fg1 is most closely related to Fm1 (sharing88.8% identity) while the bifunctional Fm1 and Mg1 are only 60.9%identical.

It is expected that this unique class of fungal Δ15 desaturases will beuseful for expression in oleaginous yeast and plants (e.g., Yarrowialipolytica) as a means to alter the fatty acid composition, based on theexpectation that they will function with high efficiency (i.e., percentsubstrate conversion, wherein % substrate conversion of LA to ALA of atleast about 50% is useful, a conversion efficiency to ALA of at leastabout 80% is preferred, a % substrate conversion to ALA of at leastabout 90% is particularly suitable, and a % substrate conversion to ALAof at least about 95% is most preferred). Thus, one embodiment of theinvention is a method of altering fatty acid profiles in an oleaginousyeast, whereby a Δ15 desaturase protein of sub-family 1 is expressedalone or in combination with other fatty acid biosynthetic genes (e.g.,a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ12 desaturase, aΔ15 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8 desaturaseand/or an elongase). A second embodiment is a method of altering fattyacid profiles in plants, whereby a whereby a Δ15 desaturase protein ofsub-family 1 is expressed alone or in combination with other fatty acidbiosynthetic genes to alter the omega3:omega6 ratios in the oils and/orto alter the accumulation or composition of plant PUFAs.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1.) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2^(nd) ed., Sinauer Associates: Sunderland,Mass. (1989). All reagents, restriction enzymes and materials used forthe growth and maintenance of bacterial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

E. coli TOP10 cells and E. coil Electromax DH10B cells were obtainedfrom Invitrogen (Carlsbad, Calif.). Max Efficiency competent cells of E.coli DH5α were obtained from GIBCO/BRL (Gaithersburg, Md.). E. coli (XL1-Blue) competent cells were purchased from the Stratagene Company (SanDiego, Calif.). All E. coli strains were typically grown at 37° C. onLuria Bertani (LB) plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). Oligonucleotides were synthesized bySigma-Genosys (Spring, Tex.). PCR products were cloned into Promega'spGEM-T-easy vector (Madison, Wis.).

DNA sequence was generated on an ABI Automatic sequencer using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). Allsequences represent coverage at least two times in both directions.Comparisons of genetic sequences were accomplished using DNASTARsoftware (DNASTAR Inc., Madison, Wis.).

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μl” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strains ATCC #76982 and ATCC #90812 were purchasedfrom the American Type Culture Collection (Rockville, Md.). Y.lipolytica strains were usually grown at 28° C. on YPD agar (1% yeastextract, 2% bactopeptone, 2% glucose, 2% agar). For transformationselection, minimal medium (0.17% yeast nitrogen base (DIFCOLaboratories, Detroit, Mich.) without ammonium sulfate and without aminoacids, 2% glucose, 0.1% proline, pH 6.1) was used. Supplements ofadenine, leucine, lysine and/or uracil were added as appropriate to afinal concentration of 0.01%.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G., and Nishida I. Arch Biochem Biophys. 276(1):3846(1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fittedwith a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oventemperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Example 1 Construction of Yarrowia Expression Vectors

The present Example describes the construction of pY5-13 (comprising achimeric TEF promoter::XPR terminator gene), pY5-13GPDN (comprising achimeric GPD promoter::XPR terminator gene), and pY5-20 (comprising achimeric hygromycin resistance gene).

Construction of Plasmid PY5-13

The plasmid pY5, a derivative of pINA532 (a gift from Dr. ClaudeGaillardin, Insitut National Agronomics, Centre de biotechnologieAgro-Industrielle, laboratoire de Genetique Moleculaire et CellularieINRA-CNRS, F-78850 Thiverval-Grignon, France), was constructed forexpression of heterologous genes in Yarrowia lipolytica (FIG. 3). First,the the partially-digested 3598 bp EcoRI fragment containing the ARS18sequence and LEU2 gene of pINA532 was subcloned into the EcoRI site ofpBluescript (Strategene, San Diego, Calif.) to generate pY2. The TEFpromoter (Muller S., et al., Yeast, 14: 12671283 (1998)) was amplifiedfrom Yarrowia lipolytica genomic DNA by PCR using TEF5′ (SEQ ID NO:21)and TEF3′ (SEQ ID NO:22) as primers. PCR amplification was carried outin a 50 μl total volume containing: 100 ng Yarrowia genomic DNA, PCRbuffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75),2 mM MgSO₄, 0.1% Triton X-100), 100 μg/mL BSA (final concentration), 200μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1μl of PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.).Amplification was carried out as follows: initial denaturation at 95° C.for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56°C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for10 min was carried out, followed by reaction termination at 4° C. The418 bp PCR product was ligated into pCR-Blunt to generate pIP-tef. TheBamHI/EcoRV fragment of pIP-tef was subcloned into the BamHI/SmaI sitesof pY2 to generate pY4.

The XPR2 transcriptional terminator was amplified by PCR using pINA532as template and XPR5′ (SEQ ID NO:23) and XPR3′ (SEQ ID NO:24) asprimers. The PCR amplification was carried out in a 50 μl total volume,using the components and conditions described above. The 179 bp PCRproduct was digested with SaclI and then ligated into the SaclI site ofpY4 to generate pY5. Thus, pY5 (shown in FIG. 3) is useful as aYarrowia-E. coli shuttle plasmid containing:

-   -   1.) a Yarrowia autonomous replication sequence (ARS18);    -   2.) a ColE1 plasmid origin of replication;    -   3.) an ampicillin-resistance gene (Amp^(R)), for selection in E.        coli;    -   4.) a Yarrowia LEU2 gene, for selection in Yarrowia;    -   5.) the translation elongation promoter (TEF P), for expression        of heterologous coding regions in Yarrowia; and    -   6.) the extracellular protease gene terminator (XPR2) for        transcriptional termination of heterologous gene expression in        Yarrowia.

Plasmid pY5-13 was constructed as a derivative of pY5 to faciliatesubcloning and heterologous gene expression in Yarrowia lipolytica.

Specifically, pY5-13 was constructed by 6 rounds of site-directedmutagenesis using pY5 as template. Both SalI and ClaI sites wereeliminated from pY5 by site-directed mutagenesis using oligonucleotidesYL5 and YL6 (SEQ ID NOs:25 and 26) to generate pY5-5. A SalI site wasintroduced into pY5-5 between the Leu2 gene and the TEF promoter bysite-directed mutagenesis using oligonucleotides YL9 and YL10 (SEQ IDNOs:27 and 28) to generate pY5-6. A PacI site was introduced into pY5-6between the LEU2 gene and ARS18 using oligonucleotides YL7 and YL8 (SEQID NOs:29 and 30) to generate pY5-8. A NcoI site was introduced intopY5-8 around the translation start codon of the TEF promoter usingoligonucleotides YL3 and YL4 (SEQ ID NOs:31 and 32) to generate pY5-9.The NcoI site inside the Leu2 gene of pY5-9 was eliminated using YL1 andYL2 oligonucleotides (SEQ ID NOs:33 and 34) to generate pY5-12. Finally,a BsiWI site was introduced into pY5-12 between the ColEI and XPR regionusing oligonucleotides YL61 and YL62 (SEQ ID NOs:35 and 36) to generatepY5-13.

Construction f Plasmid pY5-13GPDN

A DNA fragment including the glyceraldehyde-3-phosphate-dehydrogenase(GPD) promoter region (“GPDPro”; see co-pending U.S. patent applicationSer. No.10/869630, herein incorporated by reference in its entirety) wasamplified with oligonucleotides YL211 (SEQ ID NO:38) and YL212 (SEQ IDNO:39) as primers using Yarrowia genomic DNA as template. Briefly, thispromoter fragment (SEQ ID NO:37) was comprised of the nucleotide regionbetween the −968 to +3 region of the GPD gene, wherein the ‘A’ positionof the ‘ATG’ translation initiation codon is designated as +1.

The amplified GPDPro DNA fragment was completely digested with SalI andthen partially digested with NcoI. The SalI/NcoI fragment containingGPDPro was purified following gel electrophoresis in 1% (w/v) agaroseand ligated to NcoI/SalI digested pY5-13 vector (wherein the NcoI/SalIdigestion had excised the TEF promoter from the pY5-13 vector backbone)to yield pY5-13GPD. Thus, pY5-13GPD comprised a GPDPro::XPR terminatorexpression cassette.

The Nco I site at the 3′ end of the promoter fragment in pY5-13GPD wasconverted to a Not I site to yield pY5-13GPDN. For this, GPDPro wasre-amplified by PCR using GPDsense (SEQ ID NO:40) and GPDantisense (SEQID NO:41) primers with a Not I site. The resultant promoter fragment wasdigested with Sal I and Not I and cloned into the Sal/NotI site ofpY5-13 (thus removing the TEF promoter) to produce pY5-13GPDN.

Construction of Plasmid pY5-20

Plasmid pY5-20 is a derivative of pY5. It was constructed by inserting aNot I fragment containing a chimeric hygromycin resistance gene into theNot I site of pY5. The chimeric gene had the hygromycin resistance ORFunder the control of the Yarrowia lipolytica TEF promoter.

Example 2 Cloning of the Yarrowia Lipolytica Δ12 Desaturase andDisruption of the Endogenous Δ12 Desaturase Gene

Based on the fatty acid composition of Yarrowia lipolytica (ATCC #76982)which demonstrated that the organism could make LA (18:2) but not ALA(18:3), it was assumed that Y. lipolytica would likely contain gene(s)having Δ12 desaturase activity but not Δ15 desaturase activity. Thus,the present Example describes the use of degenerate PCR primers toisolate a partial coding sequence of the Yarrowia lipolytica Δ12desaturase, the use of the partial sequence to disrupt the native genein Yarrowia lipolytica, and subsequent cloning of the full-length gene.

Cloning of a Partial Putative Δ12 Desaturase Sequence from Yarrowialipolytica by PCR Using Degenerate PCR Primers

Genomic DNA was isolated from Yarrowia lipolytica (ATCC #76982) usingDNeasy Tissue Kit (Qiagen, Catalog #69504) and resuspended in kit bufferAE at a DNA concentration of 0.5 μg/μl. PCR amplifications wereperformed using the genomic DNA as template and several sets ofdegenerate primers made to amino acid sequences conserved betweendifferent Δ12 desaturases. The best results were obtained with a set ofupper and lower degenerate primers, P73 and P76, respectively, as shownin the Table below. TABLE 5 Degenerate Primers Used For Amplification OfA Partial Putative Δ12 Desaturase Degenerate Corresponding NucleotideAmino Acid Primer Set Description Sequence Sequence P73 (32)5′-TGGGTCCTGGGCCAY WVLGHECGH 26-mers GARTGYGGNCA-3′ (SEQ ID NO:43) (SEQID NO:42) P76 (64) 5′-GGTGGCCTCCTCGGC (M/I)PFVHAEEAT 30-mersGTGRTARAANGGNAT-3′ (SEQ ID NO:45) (SEQ ID NO:44)[Note:Abbreviations are standard for nucleotides and proteins. The nucleicacid degeneracy code used is as follows: R = A/G; Y = C/T; and N =A/C/G/T.]

The PCR was carried out in an Eppendorf Mastercycler Gradientthermocycler according to the manufacturer's recommendations.Amplification was carried out as follows: initial denaturation at 95° C.for 1 min, followed by 30 cycles of denaturation at 95° C. for 30 sec,annealing at 58° C. for 1 min, and elongation at 72° C. for 1 min. Afinal elongation cycle at 72° C. for 10 min was carried out, followed byreaction termination at 4° C.

The expected (ca. 740 bp) size PCR product was detected by agarose gelelectrophoresis, isolated, purified, cloned into a pTA vector(Invitrogen), and sequenced. The resultant sequence had homology toknown Δ12 desaturases, based on BLAST program analysis (Basic LocalAlignment Search Tool; Altschul, S. F., et al., J. Mol. Biol.215:403-410 (1993)).

Targeted Disruption of Yarrowia lipolytica Δ12 Desaturase Gene

Targeted disruption of the Δ12 desaturase gene in Yarrowia lipolyticaATCC #76982 was carried out by homologous recombination-mediatedreplacement of the Δ12 desaturase gene with a targeting cassettedesignated as pY23D12. pY23D12 was derived from plasmid pY5-20 (Example1).

Specifically, pY23D12 was created by inserting a Hind III/Eco RIfragment into similarly linearized pY5-20. This 642 bp fragmentconsisted of (in 5′ to 3′ orientation): 3′ homologous sequence fromposition +718 to +1031 (of the coding sequence (ORF) in SEQ ID NO:54), aBgl II restriction site, and 5′ homologous sequence from position +403to +717 (of the coding sequence (ORF) in SEQ ID NO:54). The fragment wasprepared by PCR amplification of 3′ and 5′ sequences from the 642 bp PCRproduct using sets of PCR primers P99 and P100 (SEQ ID NOs:46 and 47)and P101 and P102 (SEQ ID NOs:48 and 49), respectively.

pY23D12 was linearized by Bgl II restriction digestion and transformedinto mid-log phase Y lipolytica ATCC #76982 cells by the lithium acetatemethod according to the method of Chen, D. C. et al. (Appl MicrobiolBiotechnol. 48(2):232-235 (1997)). Briefly, Y. lipolytica was streakedonto a YPD plate and grown at 30° C. for approximately 18 hr. Severallarge loopfuls of cells were scraped from the plate and resuspended in 1mL of transformation buffer containing:

-   -   2.25 mL of 50% PEG, average MW 3350;    -   0.125 mL of 2 M Li acetate, pH 6.0;    -   0.125 mL of 2 M DTT; and    -   50 □g sheared salmon sperm DNA.

About 500 ng of plasmid DNA was incubated in 100 □l of resuspendedcells, and maintained at 39° C. for 1 hr with vortex mixing at 15 minintervals. The cells were plated onto YPD hygromycin selection platesand maintained at 30° C. for 2 to 3 days.

Four hygromycin-resistant colonies were isolated and screened fortargeted disruption by PCR. One set of PCR primers (P119 [SEQ ID NO:50]and P120 [SEQ ID NO:51]) was designed to amplify a specific junctionfragment following homologous recombination. Another set of PCR primers(P121 [SEQ ID NO:52] and P122 [SEQ ID NO:53]) was designed to detect thenative gene. Three of the four hygromycin-resistant colonies werepositive for the junction fragment and negative for the native fragment,thus confirming targeted integration.

Determination of Fatty Acid Profile in the Δ12 Desaturase-DisruptedStrain

Disruption of the native Δ12 desaturase gene was further confirmed by GCanalysis of the total lipids in one of the disrupted strains, designatedas Q-d12D. Single colonies of wild type (ATCC #76982) and Q-d12D wereeach grown in 3 mL minimal media (formulation/L: 20 g glucose, 1.7 gyeast nitrogen base, 1 g L-proline, 0.1 g L-adenine, 0.1 g L-lysine, pH6.1) at 30° C. to an OD₆₀₀˜1.0. The cells were harvested, washed indistilled water, speed vacuum dried and subjected to directtrans-esterification and GC analysis (as described in the GeneralMethods).

The fatty acid profile of wildtype Yarrowia and the transformant Q-d12Dcomprising the disrupted Δ12 desaturase are shown below in Table 6.Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0, 18:1 (oleic acid) and 18:2 (LA) and the composition of each ispresented as a % of the total fatty acids. TABLE 6 Fatty AcidComposition (% Of Total Fatty Acids) In Wildtype And TransformantYarrowia lipolytica Strain 16:0 16:1 18:0 18:1 18:2 Wild type 11 14 2 3334 Q-d12D 6 15 1 74 nd disrupted*nd = not detectableResults indicated that the native Δ12 desaturase gene in the Q-d12Dstrain was inactivated. Thus, there is only one gene encoding afunctional Δ12 desaturase in Yarrowia lipolytica ATCC #76982.

Plasmid Rescue of the Yarrowia lipolytica Δ12 Desaturase Gene

Since the Δ12 desaturase gene was disrupted by the insertion of theentire pY23D12 vector that also contained an E. coliampicillin-resistant gene and E. coli ori, it was possible to rescue theflanking sequences in E. coli. For this, genomic DNA of Yarrowialipolytica strain Q-d12D was isolated using the DNeasy Tissue Kit.Specifically, 10 μg of the genomic DNA was digested with 50 μl ofrestriction enzymes Age I, Avr II, Nhe I and Sph I in a reaction volumeof 200 μl. Digested DNA was extracted with phenol:chloroform andresuspended in 40 μl deionized water. The digested DNA (10 μl) wasself-ligated in 200 μl ligation mixture containing 3 U T4 DNA ligase.Ligation was carried out at 16° C. for 12 hrs. The ligated DNA wasextracted with phenol:chloroform and resuspended in 40 μl deionizedwater. Finally, 1 μl of the resuspended ligated DNA was used totransform E. coli by electroporation and plated onto LB platescontaining ampicillin (Ap). Ap-resistant colonies were isolated andanalyzed for the presence of plasmids by miniprep. The following insertsizes were found in the recovered or rescued plasmids (Table 7): TABLE 7Insert Sizes Of Recovered Plasmids, According To Restriction EnzymeEnzyme plasmid insert size (kB) AgeI 1.6 AvrII 2.5 NheI 9.4 SphI 6.6Sequencing of the plasmids was initiated with sequencing primers P99(SEQ ID NO:46) and P102 (SEQ ID NO:49).

Based on the sequencing results, a full-length gene encoding theYarrowia lipolytica Δ12 desaturase gene was assembled (1936 bp; SEQ IDNO:54). Specifically, the sequence encoded an open reading frame of 1257bases (nucleotides +283 to +1539 of SEQ ID NO:54), while the deducedamino acid sequence was 419 residues in length (SEQ ID NO:55). This genewas also also publically disclosed as YALI-CDS3053.1 within the publicY. lipolytica protein database of the “Yeast project Genolevures”(Center for Bioinformatics, LaBRI, Talence Cedex, France) (see alsoDujon, B. et al., Nature 430 (6995):35-44 (2004)).

Example 3 Identification of Δ15 Desaturases from Filamentous Fungi

The present Example describes the identification of Δ15 desaturases invarious filamentous fungi. These sequences were identified based ontheir homology to the Yarrowia lipolytica Δ12 desaturase (Example 2);and, the sequences from each species fell into one of two “sub-families”based on phylogenetic analyses.

Homology Searches With Synechochytis Δ15 Desaturase

First, public databases of the filamentous fungi Neurospora crassa andMagnaporthe grisea sequences were subjected to BLAST searches (BasicLocal Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol.215:403-410 (1993)) using the Synechochytis Δ15 desaturase proteinsequence (gene desB; GenBank Accession No. D90913) as the querysequence. Unexpectedly, these searches failed to identify any homologoussequence.

Homology Searches with Yarrowia lipolytica Δ12 Desaturase

Applicants then performed BLAST searches of the same databases with theYarrowia lipolytica Δ12 desaturase protein sequence as the querysequence (SEQ ID NO:55). These searches resulted in the identificationof two homologous sequences within each organism. Subsequently, SEQ IDNO:55 was used as a query against: 1.) public databases of Aspergillusnidulans and Fusarium graminearium; and 2.) a DuPont EST library ofFusarium moniliforme strain M-8114 (E. I. duPont de Nemours and Co.,Inc., Wilmington, Del.) (F. moniliforme strain M-8114 available from theFusarium Research Center, University Park, Pa.; see also Plant Disease81(2): 211-216 (1997)). These searches also resulted in theidentification of two homologs to the Yarrowia lipolytica Δ12 desaturaseprotein within each organism. The Table below summarizes detailsconcerning each of these homologs. TABLE 8 Description Of ORFs HavingHomology To The Yarrowia lipolytica Δ12 Desaturase SEQ ID Abbre- NOs*Source viation Organism 1, 2 EST sequence database, E. I. Fm 1 FusariumduPont de Nemours and Co., Inc. moniliforme 3, 4 EST sequence database,E. I. Fm 2 Fusarium duPont de Nemours and Co., Inc. moniliforme 5, 6Contig 1.122 (scaffold 9) in the A. An1 Aspergillus nidulans genomeproject (sponsored nidulans by the Center for Genome Research (CGR),Cambridge, MA); see also WO 2003/099216 7, 8 Contig 1.15 (scaffold 1) inthe A. An2 Aspergillus nidulans genome project; nidulans AAG36933 9, 10Locus MG08474.1 in contig 2.1597 Mg1 Magnaporthe in the M. grisea genomeproject grisea (sponsored by the CGR and International Rice Blast GenomeConsortium) 11, 12 Locus MG01985.1 in contig 2.375 in Mg2 Magnaporthethe M. grisea genome project grisea 13, 14 GenBank Accession No. Nc1Neurospora AABX01000577); see also crassa WO 2003/099216 15, 16 GenBankAccession No. Nc2 Neurospora AABX01000374 crassa 17, 18 Contig 1.320 inthe F. graminearium Fg1 Fusarium genome project (sponsored by thegraminearium CGR and the International Gibberella zeae GenomicsConsortium (IGGR); BAA33772.1) 19, 20 Contig 1.233 in the F.graminearium Fg2 Fusarium genome project graminearium*Note:Odd SEQ ID NOs refer to ORF nucleotide sequences and even SEQ ID NOsrefer to the deduced amino acid sequences.

All of the homologs were either unannotated or annotated as a Δ12desaturase or fatty acid desaturase. Furthermore, the nucleotidesequences from F. graminearium were genomic with putative intronsequences; the Applicants made a tentative assembly of the deduced aminoacids for comparison with amino acid sequences from the other homologs.

Phylogenetic tree analysis of the Δ12 desaturase homologs from eachspecies using the Megalign program of the LASERGENE bioinformaticscomputing suite (Windows 32 Megalign 5.06 1993-2003; DNASTAR Inc.,Madison, Wis.) unexpectedly revealed two sub-families. As shown in FIG.4, Ncd, Mg1, Fg1, Fm1 and An1 clustered in “sub-family 1” of theproteins having homology to the Yarrowia lipolytica Δ12 desaturase whileFg2, Fm2, Mg2, Nc2 and An2 clustered within “sub-family 2” of theYarrowia lipolytica Δ12 desaturase protein homologs.

Each of the proteins having homology to the Yarrowia lipolytica Δ12desaturase were then aligned using the method of Clustal W (slow,accurate, Gonnet option; Thompson et al., Nucleic Acids Res.22:4673-4680 (1994)) of the Megalign program of DNASTAR software. Thepercent identities revealed by this method were used to determinewhether the proteins were orthologs (FIG. 5). Specifically, the FIG.shows: 1.) the percent identity among the proteins clustered withinsub-family 1 (upper left-hand corner triangle, shown with a dark line);2.) the percent identity between proteins in sub-family 1 and sub-family2 (upper right-hand corner box, shown with a dotted line); and 3.) thepercent identity among the proteins clustered within sub-family 2 (lowerright-hand corner triangle). Thus, all proteins of sub-family 1 (SEQ IDNOs:2, 6,10, 14 and 18) were at least 46.2% identical to each other andwere less than 39.6% identical to the proteins of sub-family 2 (SEQ IDNOs:4, 8, 12, 16 and 20). Furthermore, the proteins of sub-family 2 wereat least 56.3% identical to each other.

The analyses above clearly differentiated the two sub-families ofproteins having homology to the Yarrowia lipolytica Δ12 desaturase (SEQID NO:55). Additionally, it was known that yeast such as Y. lipolyticacan only synthesize 18:2 (but not 18:3), while each of the fivefilamentous fungi are able to synthesize both 18:2 and 18:3.Furthermore, a single Δ12 desaturase was isolated from Yarrowia, whileall of the fungi had two homologs to theYarrowia Δ12 desaturase. Thus,the Applicants postulated that one of the sub-families of desaturases inthese organisms represented a Δ12 desaturase (permitting conversion ofoleic acid to LA (18:2)) and the other represented a Δ15 desaturase(permitting conversion of LA to ALA (18:3)).

Finally, the Fusarium moniliforme Δ15 desaturase protein sequence wasanalyzed individually for its similarity using a ClustalW alignmentalgorithm (Megalign program of DNASTAR software, supra) to known Δ15desaturase proteins from a wide range of species. The Fm1 amino acidsequence reported herein shares 25.4% identity with C. elegans GenBankAccession No. L41807, 33.1% identity with Synechosystis desB (GI1653388), 33.7% identity with the Arabidopsis thaliana fad2 gene, and29.1 % identity with the Saprolegnia diclina desaturase of U.S.2003/0196217.

Example 4 Construction of Expression Plasmid PY34 (GPDPro::Fm1::XPR),Comprising the Fusarium moniliforme Desaturase of Sub-Family 1 (Encodinga Putative Δ15 Desaturase)

The present Example describes the construction of an expression plasmidcomprising the Fusarium Moniliforme Δ15 desaturase of sub-family 1(“Fm1”) identified in Example 3. Specifically, a chimeric gene wascreated, such that the putative Δ15 desaturase would be expressed underthe control of the Yarrowia GPD promoter (“GPDPro”). This would enablesubsequent determination of the protein's activity in Yarrowialipolytica, by testing the ability of the expressed ORF to confer ALAproduction in the wild type strain and to complement a Δ12desaturase-disrupted mutant (Example 2).

The ORF encoding the F. moniliforme Δ15 desaturase was PCR amplifiedusing the cDNA clones ffm1c.pK001.g23 and ffm1c.pK013.n7 containing thefull-length cDNA as the template and using upper and lower primers P192(SEQ ID NO:56) and P193 (SEQ ID NO:57). The PCR was carried out in anEppendorf Mastercycler Gradient Cycler using pfu polymerase, per themanufacturer's recommendations. Amplification was carried out asfollows: initial denaturation at 95° C. for 1 min, followed by 30 cyclesof denaturation at 95° C. for 30 sec, annealing at 58° C. for 1 min andelongation at 72° C. for 1 min. A final elongation cycle at 72° C. for10 min was carried out, followed by reaction termination at 4° C.

The correct-sized (ca. 1240 bp) fragment was obtained from bothtemplates. The fragment derived from clone ffm1c.pK001.g23 was purifiedfrom an agarose gel using a Qiagen DNA purification kit (Valencia,Calif.), digested with Not I and cloned into the Not I site betweenGPDPro and the XPR terminator of plasmid pY5-13GPDN (from Example 1).This resulted in creation of plasmid pY34, which contained aGPDPro::Fm1::XPR chimeric gene. The sequence of the Fm1 ORF in theresultant 8878 bp plasmid was confirmed. Plasmid pY34 additionallycontained the E. coli origin of replication, the bacterial ampicillinresistance gene, a Yarrowia Leu 2 gene and the Yarrowia autonomousreplication sequence (ARS).

Example 5 Expression of Plasmid PY34 (GPDPro::Fm1::XPR), Comprising theFusarium moniliforme Desaturase of Sub-Family 1 (Encoding a Putative Δ15Desaturase) un Yarrowia lipolytica

The present Example describes expression of plasmid pY34 (comprising thechimeric GPDPro::Fm1::XPR gene; from Example 4) in Yarrowia lipolytica.Specifically, the ability of the expressed F. moniliforme ORF to conferALA production in the wild type strain of Y. lipolytica (therebyconfirming the ORF's Δ15 desaturase activity) and to complement the Δ12desaturase-disrupted mutant (from Example 2; thereby confirming theORF's bifunctional Δ12/Δ15 desaturase activity) was tested.

Plasmids pY5 (vector alone control, from Example 1) and pY34(GPDPro::Fm1::XPR) were each individually transformed into wild type(WT) and Δ12 desaturase-disrupted (Q-d12D) strains of Yarrowialipolytica ATCC #76892, using the transformation procedure described inExample 2. Transformant cells were selected on Bio101 DOB/CSM-Leuplates.

Single colonies of wild type and transformant cells were each grown in 3mL minimal media, harvested, washed, dried and analyzed, as described inExample 2 and the General Methods.

The fatty acid profile of wildtype Yarrowia and each of thetransformants are shown below in Table 9. Fatty acids are identified as16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2(LA) and 18:3 (ALA) and the composition of each is presented as a % ofthe total fatty acids. “d12d % SC” was calculated according to thefollowing formula: ([18:2+18:3]/[18:1+18:2+18:3])*100 and representspercent substrate conversion to 18:2. “d15d % SC” was calculatedaccording to the following formula: ([18:3]/[18:2+18:3])*100 andrepresents percent substrate conversion to ALA. TABLE 9 IdentificationOf The Fusarium moniliforme Fm1 As A Bifunctional Δ12/Δ15 Desaturase % %% % % % d12d d15d Ratio Strain 16:0 16:1 18:0 18:1 18:2 ALA % SC % SCALA/LA WT 12.1 9.1 0.8 33.8 44.2 0.0 56.7 0 — WT + GPDPro:: 10.0 10.51.3 37.0 7.2 31.0 52.6 82.5 4.3 Fm1::XPR Q-d12D 3.3 13.9 0.3 82.4 0.00.0 0.0 — — Q-d12D + GPDPro:: 7.8 12.0 1.0 60.4 0.7 17.8 23.7 96.3 25.2Fm1::XPR

The results above demonstrated that the F. moniliforme ORF referred toherein as Fm1, and identified as a protein within sub-family 1 of thoseproteins having homology to the Yarrowia lipolytica Δ12 desaturase, is aΔ15 desaturase. Based on this confirmation, the Applicants predict thatall other members of sub-family 1 (SEQ ID NOs:6, 10, 14 and 18) alsowill have Δ15 desaturase functionality.

Concerning the Δ15 desaturase activity of Fm1, it is noteworthy that theprotein is even more efficient in its activity in Yarrowia (31% ALAaccumulation) than previously expressed Δ15 desaturases in other yeast.Specifically, the % product accumulation of ALA when expressing the C.elegans Δ15 desaturase in the non-oleaginous yeast Saccharomycescerevisiae was only 4.1 % (Meesapyodsuk et al., Biochem. 39:11948-11954(2000)), while the % product accumulation of ALA when expressing the B.napus Δ15 desaturase in S. cerevisiae was only 1.3% (Reed., D. W. etal., Plant Physiol. 122:715-720 (2000)). Based on the results providedherein, it would be expected that expression of the Fusarium moniliformeΔ15 desaturase, in combination of other genes for PUFA biosynthesis(e.g., a Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase, Δ9desaturase, Δ8 desaturase, Δ4 desaturase, Δ12 desaturase), would resultin higher production of ω-3 PUFAs than would result using any of thepreviously identified Δ15 desaturases.

Additionally, the results demonstrated that, unexpectedly, the Fusariummoniliforme Δ15 desaturase (Fm1) has some Δ12 desaturase activity.Specifically, expression of Fm1 in the Δ12 desaturase-disrupted strainof Yarrowia lipolytica (i.e., Q-d12D +GPDPro::Fm1::XPR) resulted in 24%substrate conversion of oleic acid to LA due to the Δ12 desaturasefunctionality of Fm1 (see “d12d % SC”). This was in addition to highsubstrate conversion of LA to ALA (96%, see “d15d % SC”) due to the Δ15desaturase functionality of Fm1. This bifunctionality is in markedcontrast to any other known Δ12 or Δ15 desaturase. It will be obvious toone of skill in the art that expression of the Fusarium moniliforme Δ15desaturase in a host organism that has low Δ12 desaturase activity (orlacks such activity entirely) will lead to maximized ratios of18:3/18:2. It would be expected that when other genes for PUFAbiosynthesis (e.g., a Δ6 desaturase, elongase, Δ5 desaturase, Δ17desaturase) were expressed in this type of host organism with theFusarium moniliforme Δ15 desaturase described above, an increased ratioof ω-3 to ω-6 fatty acids would result.

Example 6 Expression of Magnaporthe grisea Desaturase of Sub-Family 1(Encoding a Putative Δ15 Desaturase) in Yarrowia lipolytica

The present Example describes the construction of an expression plasmidcomprising the putative Magnaporthe grisea Δ15 desaturase (“Mg1”) andthe expression of this plasmid in Yarrowia lipolytica. This enabledconfirmation of Mg1 as a Δ15 desaturase by testing the ability of theexpressed ORF to confer ALA production in the wild type Yarrowialipolytica strain and as a bifunctional Δ12/Δ15 desaturase by testingthe ability of the expressed ORF to confer ALA production in the Δ12desaturase-disrupted mutant of Yarrowia lipolytica (from Example 2).

Specifically, a chimeric TEF::Mg1 gene was constructed, wherein theputative Δ15 desaturase was expressed under the control of a YarrowiaTEF promoter (Muller S., et al., Yeast, 14: 12671283 (1998)). First,Magnaporthe grisea genomic DNA was isolated in a manner similar to thatdescribed for Yarrowia lipolytica in Example 2. Then, since theMagnaporthe grisea Mg1 gene encoding the putative Δ15 desaturase (SEQ IDNO:9) has two introns, these sequences were removed during PCRamplification by first PCR-amplifying the three exons separately, andthen PCR-amplifying the full length ORF by joining the three exonstogether using overlapping PCR primers. Thus, genomic DNA was used asthe template in 3 separate PCR reactions, using the upper and lowerprimers shown below in Table 10. TABLE 10 Primers For Amplification OfMagnaporthe grisea Exons Encoding Mg1 Exon to be Amplified Upper PrimerLower Primer Exon 1 P186 (SEQ ID NO: 59) P187 (SEQ ID NO: 60) Exon 2P188 (SEQ ID NO: 61) P189 (SEQ ID NO: 62) Exon 3 P190 (SEQ ID NO: 63)P191 (SEQ ID NO: 64)

Then, the full-length ORF was PCR-amplified using all three gel purifiedPCR products as templates and upper primer P186 and lower primer P191.Primers P186 and P191 contained Not I sites to facilitate cloning intothe expression vector. Specifically, the correct-sized fragment wasgel-purified, digested with NcoI and Not I, and cloned into Not I—cutYarrowia expression vector pY5-13 (Example 1) under the control of theYarrowia TEF promoter. The resultant clones were designated pY31.

Several pY31 clones were sequenced. As expected, all had a T-to-Csubstitution at position 3 of the ORF due to the NcoI site that wascreated in the upper primer to facilitate the cloning. This resulted ina change in the second amino acid from Ser to Ala. Three plasmid clones(i.e., pY31 plasmid clones #21, #24 and #28) were encoded by SEQ IDNO:10 (except for the second amino acid change described above);however, none of them had a nucleotide sequence identical to that of SEQID NO:9 (i.e., they had additional silent nucleotide substitutions thatdid not change the deduced amino acid sequence and most likely occurredby PCR errors). More specifically, plasmid clones #21, #24 and #28 allhad the following base substitutions: a C-to-T substitution at position309, a C-to-T substitution at position 390, a T-to-C substitution atposition 549 and a G-to-C substitution at position 567. In addition,clone #24 had a T-to-A substitution at position 645, and both clones #21and #28 had a C-to-T substitution at position 669.

The plasmids comprising the TEF::Mg1 chimeric genes (i.e., clones #21,#24 and #28) were each transformed into wildtype (Q) and the Δ12desaturase-disrupted strain (Q-d12D) of Yarrowia lipolytica (ATCC#76982), according to the methodology described in Example 2. Threecolonies (identified as “a”, “b” and “c” in Table 11 below) from eachtransformation were picked and inoculated into 3 mL DOB/CSM medium andgrown at 30° C. for 72 hrs, as described in General Methods. Cultures(1.5 mL) were harvested and subjected to direct trans-esterification andGC analysis (as described in Example 2 and the General Methods).

The fatty acid profile of wildtype Yarrowia and each of thetransformants are shown below in Table 11. Fatty acids are identified as16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2(LA) and 18:3 (ALA) and the composition of each is presented as a % ofthe total fatty acids (TFAs). “d12d % SC” was calculated according tothe following formula: ([18:2+18:3]/ [18:1+18:2+18:3])*100 andrepresents percent substrate conversion to 18:2. “d15d % SC” wascalculated according to the following formula: ([18:3]/[18:2+18:3])*100and represents percent substrate conversion to ALA. TABLE 11Identification Of The Magnaporthe grisea Mg1 As A Bifunctional Δ12/Δ15Desaturase Plasmid, TFA % % % % % % d12d d15d Strain Transformant □g)16:0 16:1 18:0 18:1 18:2 ALA % SC % SC Q-D12 None 341 4.2 10.8 1.4 80.40.0 0.0 0 Q-D12 pY31#21, a 283 5.1 13.5 1.5 75.8 0.0 1.3 2 100 Q-D12pY31#21, b 257 5.1 13.2 1.4 76.0 0.0 1.4 2 100 Q-D12 pY31#21, c 255 5.213.0 1.5 76.0 0.0 1.4 2 100 Q-D12 pY31#24, a 261 5.1 13.6 1.5 75.5 0.01.3 2 100 Q-D12 pY31#24, b 272 5.0 13.0 1.4 76.0 0.0 1.4 2 100 Q-D12pY31#24, c 321 5.3 12.7 1.4 76.0 0.0 1.6 2 100 Q-D12 pY31#28, a 289 5.013.3 1.4 76.0 0.0 1.4 2 100 Q-D12 pY31#28, b 317 5.0 13.3 1.4 76.1 0.01.3 2 100 Q-D12 pY31#28, c 284 5.1 13.3 1.5 75.9 0.0 1.4 2 100 Q None258 7.1 13.0 1.3 46.6 29.2 0.0 39 0 Q pY31#21, a 243 6.4 14.2 1.2 50.811.5 13.4 33 54 Q pY31#21, b 297 6.4 14.0 1.3 51.0 11.5 13.4 33 54 QpY31#21, c 269 6.5 14.1 1.3 51.0 11.3 13.2 32 54 Q pY31#24, a 240 6.613.9 1.4 50.8 10.9 14.0 33 56 Q pY31#24, b 249 6.6 14.1 1.4 51.0 11.113.3 32 55 Q pY31#24, c 219 6.5 14.1 1.4 50.9 11.2 13.4 33 55 Q pY31#28,a 311 6.3 14.2 1.2 51.4 10.9 13.5 32 55 Q pY31#28, b 296 6.0 14.1 1.251.7 11.0 13.6 32 55 Q pY31#28, c 264 6.3 14.2 1.3 51.6 10.9 13.2 32 55

As shown above, ALA is produced in both wildtype (Q) and Δ12desaturase-disrupted strains (Q-d12D) of Yarrowia lipolytica that weretransformed with the TEF::Mg1 chimeric gene. Thus, on the basis of theseresults, the identify of Mg1 as a desaturase having bifunctional Δ12/Δ15activity is confirmed.

Example 7 Expression of Fusarium graminearium Desaturase of Sub-Family 1(Encoding a Putative Δ15 Desaturase) in Yarrowia lipolytica

The present Example describes the construction of an expression plasmidcomprising the putative Fusarium gramine Δ15 desaturase (“Fg1”) and theexpression of this plasmid in Yarrowia lipolytica. This would enableconfirmation of Fg1 as a Δ15 desaturase by testing the ability of theexpressed ORF to confer ALA production in the wild type Yarrowialipolytica strain and as a bifunctional Δ12/Δ15 desaturase by testingthe ability of the expressed ORF to confer ALA production in the Δ12desaturase-disrupted mutant of Yarrowia lipolytica (from Example 2).

Specifically, a chimeric TEF::Fg1 gene will be synthesized, wherein theputative Δ15 desaturase would be expressed under the control of aYarrowia TEF promoter. In a manner similar to that described in Example6, three introns present in the Fusarium graminearium Fg1 gene encodingthe putative Δ15 desaturase (SEQ ID NO:17) will be removed during PCRamplification, prior to expression of the Fg1 ORF. Thus, genomic F.graminearium DNA will first be used as template in 4 separate PCRreactions using the upper and lower primers shown below in Table 12.TABLE 12 Primers For Amplification Of F. graminearium Exons Encoding Fg1Exon to be Amplified Upper Primer Lower Primer Exon 1 PFg1UP1 (SEQ IDNO: 65) PFg1LP1 (SEQ ID NO: 66) Exon 2 PFg1UP2 (SEQ ID NO: 67) PFg1LP2(SEQ ID NO: 68) Exon 3 PFg1UP3 (SEQ ID NO: 69) PFg1LP3 (SEQ ID NO: 70)Exon 4 PFg1UP4 (SEQ ID NO: 71) PFg1LP4 (SEQ ID NO: 72)

Then, the full-length ORF will be PCR-amplified using all 4 gel purifiedPCR products as templates and upper primer PFg1 UP1 and lower primerPFg1 LP4. Primers PFg1 UP1 and PFg1 LP4 contain Not I sites tofacilitate cloning into the expression vector. The correct-sizedfragment will be gel-purified, digested with Not I, and cloned into aNot I cut Yarrowia expression vector, as described in Example 6 for theMg1 coding region.

The plasmid comprising the TEF::Fg1 chimeric genes will be transformedinto wild type (Q) and the Δ12 desaturase-disrupted (Q-d12D) strains ofYarrowia lipolytica (ATCC #76982). Following growth of single coloniesof wild type and transformant cells in minimal media, the cells will beharvested and subjected to direct trans-esterification and GC analysis(as described in Example 2 and the General Methods).

ALA is expected to be produced in both wildtype and Δ12desaturase-disrupted strains of Yarrowia lipolytica that weretransformed with the TEF::Fg1 chimeric gene, thus confirming theidentify of Fg1 as a bifunctional Δ12/Δ15 desaturase. This expectationis based on Fg1 protein sequence being closest to that of Fm1, based ona % identity comparison.

Example 8 Transformation of Arabidopsis Plants with a Chimeric GeneComprising the Fusarium moniliforme Δ15 Desaturase of Sub-Family 1 (Fm1)

This Example describes methods that will be useful to transform wildtype and fad2-1 mutant Arabidopsis with a chimeric gene containing theFusarium Δ15 desaturase coding region.

Construction of an Arabidopsis Expression Vector Comprising Fm1

The NotI fragment of pY34 containing the Fm1 Δ15 desaturase codingregion (Example 4) will be cloned into the NotI site of soybeanexpression vector pKR353. As described in example 18 below, pKR353contains a NotI site flanked by a seed-specific promoter from the Kti3(kunitz trypsin inhibitor 3) gene and a Kti3 transcription terminator.

The chimeric Kti3 promoter::Fm1::Kti3 terminator gene will be isolatedas an Asc 1 fragment from pKR353(Δ15) and cloned into the unique Asc 1site in the binary vector pZBL11 (Asc1). pZBL11 (AscI) was derived frombinary vector pZBL11 by adding an Asc I linker between the Pac I andAsp718 sites between the right and left T-DNA borders. pZBL11 (U.S. Pat.No. 5,968,793; EP 1003891; and WO 9859062) also contains a35S:sulfonylurea resistant acetolactate synthase (ALS) transgene withinthe T-DNA borders that confers resistance to sulfonylurea herbicide andserves as the plant selectable marker. pZBL11 also has an origin ofreplication of both E. coli and Agrobacterium tumefaciens, and abacterial ampicillin resistance gene.

The resultant binary plasmid, pZBLI(Δ15) will be transformed intoAgrobacterium strain LBA4404. The transfected Agrobacterium willsubsequently be used to transform wild type and a fad2-1 mutant (Okuley,J. et al., Plant Cell 6:147-158 (1994)) of Arabidopsis thaliana by theAgrobacterium dip method. Transformants will be selected on sulfonylureaand tested for ALA production in the seeds.

Wild type Arabidopsis transformed with the chimeric gene expressing theΔ15 desaturase will contain higher ALA content than untransformedplants. Fad2-1 plants transformed with the same chimeric gene willcontain a higher ALA content than the untransformed mutant; andtransformant fad2-1 plants will have a ratio of 18:3/18:2 that is higherthan in the untransformed mutant as well as the wild-type transformant.

Example 9 Transformation of Somatic Soybean Embryo Cultures with aChimeric Gene Comprising the Fusarium moniliforme Δ15 Desaturase ofSub-Family 1 (Fm1)

This Example describes methods that will be used for the cultivation ofsoybean, following their transformation with a chimeric gene containingthe Fm1 Δ15 desaturase coding region.

Construction of a Soybean Expression Vector Comprising Fm1

Plasmid pKR353(A15) (created in Example 8) will also contain twocassettes comprising the hygromycin B phosphotransferase gene (“hpt”;Gritz, L. and J. Davies, Gene 25:179-188 (1983)):

(1) A T7 promoter::hpt::T7 terminator cassette—this cassette, inaddition to a bacterial origin of replication (ori), enabled selectionand replication in E. coli.

(2) A 35S promoter::hpt::NOS 3′ transcription terminator cassette—thiscassette enabled selection in soybean (35S, see Odell et al., Nature313:810-812 (1985)); NOS 3′, see Depicker et al., J. Mol. Appl. Genet.1:561:570 (1982)).

Transformation of Somatic Soybean Embryo Cultures

Soybean embryogenic suspension cultures (cv. Jack) will be maintained in35 mL liquid medium SB196 (infra) on a rotary shaker at 150 rpm and 26°C. with cool white fluorescent lights on a 16:8 hr day/night photoperiodwith a light intensity of 60-85 μE/m²/s. SB 196 - FN Lite LiquidProliferation Medium (Per Liter): MS FeEDTA 100× Stock 10 mL MS Sulfate100× Stock 10 mL FN Lite Halides 100× Stock 10 mL FN Lite P, B, Mo 100×Stock 10 mL B5 vitamins (1 mL/L) 1.0 mL 2,4-D (10 mg/L finalconcentration) 1.0 mL KNO₃ 2.83 g (NH₄)₂SO₄ 0.463 g Asparagine 1.0 gSucrose (1%) 10 g pH 5.8

FN Lite Stock Solutions 1000 mL 500 mL 1 MS Fe EDTA 100× Stock Na₂ EDTA*3.724 g 1.862 g FeSO₄—7H₂O 2.784 g 1.392 g 2 MS Sulfate 100× stockMgSO₄—7H₂O 37.0 g 18.5 g MnSO₄—H₂O 1.69 g 0.845 g ZnSO₄—7H₂O 0.86 g 0.43g CuSO₄—5H₂O 0.0025 g 0.00125 g 3 FN Lite Halides 100× Stock CaCl₂—2H₂O30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl₂—6H₂O 0.0025 g 0.00125 g 4 FNLite P, B, Mo 100× Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 gNa₂MoO₄—2H₂O 0.025 g 0.0125 g*Add first, dissolve in dark bottle while stirring

Cultures will be subcultured every 7-14 days by inoculatingapproximately 35 mg of tissue into 35 mL of fresh liquid SB196 (thepreferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures will be transformed withpKR353(A15) (supra) by the method of particle gun bombardment (Klein etal., Nature, 327:70 (1987)). A DuPont Biolistic PDS1000/HE instrument(helium retrofit) will be used for all transformations (E.I. duPont deNemours and Co., Inc., Wilmington, Del.).

Soybean Embryogenic Suspension Culture Initiation

Soybean cultures will be initiated twice each month with 5-7 daysbetween each initiation.

Between 45-55 days after planting, pods with immature seeds fromavailable soybean plants will be picked and the seeds will be removedfrom their shells and placed into a sterilized magenta box. The soybeanseeds will be sterilized by shaking for 15 min in the followingsolution: 95 mL of autoclaved distilled water plus 5 mL Clorox and 1drop of soap. Seeds will be rinsed using two 1 L bottles of steriledistilled water and those less than 4 mm will be placed on individualmicroscope slides. The small end of the seed will be cut and thecotyledons pressed out of the seed coat. Cotyledons (25-30 per plate)will be transferred to plates containing SB1 medium.

SB1 Solid Medium (Per Liter):

1 package MS salts (Gibco/BRL, Catalog #11117-066)

1 mL B5 Vitamins Stock (infra)

31.5 g sucrose

2 mL 2,4-D (20 mg/L final concentration; 2,4-D stock is obtained premadefrom Phytotech, Catalog #D 295 as 1 mg/mL)

pH to 5.7

8 g TC agar

B5 Vitamins Stock (Per 100 mL):

10 g myo-inositol

100 mg nicotinic acid

100 mg pyridoxine HCl

1 g thiamine

* Note: Store aliquots at −20° C.; If the solution does not dissolvequickly enough, apply a low level of heat via the hot stir plate.

Plates containing the cotyledons will be wrapped with fiber tape andstored for 8 wks. After this time, secondary embryos will be cut andplaced into SB196 liquid media for 7 days.

Preparation of DNA For Bombardment

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene will be used for bombardment.Fragments are obtained by gel isolation of double digested plasmids. Ineach case, 100 μg of plasmid DNA is digested in 0.5 mL of theappropriate enzyme mix. The resulting DNA fragments are separated by gelelectrophoresis on 1% SeaPlaque GTG agarose (BioWhittaker MolecularApplications, Rockland, Me.) and the DNA fragments containing chimericgenes are cut from the agarose gel. DNA is purified from the agaroseusing the GELase digesting enzyme following the manufacturer's protocol(EpiCentre, Madison, Wis.).

A 50 μl aliquot of sterile distilled water containing 3 mg of goldparticles (3 mg gold) will be added to 5 μl of a 1 μg/μl DNA solution(either intact plasmid or DNA fragment prepared as described above), 50μl 2.5 M CaCl₂ and 20 μl of 0.1 M spermidine. The mixture will be shaken3 min on level 3 of a vortex shaker and spun for 10 sec in a benchmicrofuge. After a wash with 400 μl 1100% ethanol, the pellet will besuspended by sonication in 40 μl of 100% ethanol. Five μl of DNAsuspension will be dispensed to each flying disk of the BiolisticPDS1000/HE instrument disk. Each 5 μl aliquot will contain approximately0.375 mg gold per bombardment (i.e., per disk).

Tissue Preparation and Bombardment with DNA

Approximately 150-200 mg of 7 day old embryonic suspension cultures willbe placed in an empty, sterile 60×15 mm petri dish and the dish will becovered with plastic mesh. Tissue will be bombarded 1 or 2 shots perplate with the membrane rupture pressure set at 1100 PSI and the chamberevacuated to a vacuum of 27-28 inches of mercury. Tissue will be placedapproximately 3.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos

Transformed embryos will be selected using hygromycin (when thehygromycin phosphotransferase, HPT, gene is used as the selectablemarker) or chlorsulfuron (when the acetolactate synthase, ALS, gene isused as the selectable marker). In either case, the tissue will beplaced into fresh SB196 media and cultured as described above followingbombardment. Six days post-bombardment, the SB196 will be exchanged withfresh SB196 containing a selection agent of either 30 mg/L hygromycin or100 ng/mL chlorsulfuron (chlorsulfuron stock: 1 mg/mL in 0.01 N ammoniumhydroxide). The selection media will be refreshed weekly. Four to sixweeks post-selection, green, transformed tissue may be observed growingfrom untransformed, necrotic embryogenic clusters. Isolated, greentissue will be removed and inoculated into multiwell plates containingSB196 to generate new, clonally propagated, transformed embryogenicsuspension cultures.

Regeneration of Soybean Somatic Embryos into Plants

In order to obtain whole plants from embryogenic suspension cultures,the tissue must be regenerated.

Embryo Maturation: Embryos will be cultured for 4-6 wks at 26° C. inSB196 under cool white fluorescent (Phillips Cool White EconowattF40/CW/RS/EW) and Agro (Phillips F40 Agro; 40 watt) bulbs on a 16:8 hrphotoperiod with a light intensity of 90120 μE/m²/s. After this time,embryo clusters will be removed to SB166 solid agar media for 1-2 weeks.

SB 166 Solid Medium (Per Liter):

1 package MS salts (Gibco/BRL, Cat# 11117-066)

1 mL B5 vitamins 1000× stock

60 g maltose

750 mg MgCl₂ hexahydrate

5 g activated charcoal

pH 5.7

2 g gelrite

Clusters are then subcultured to medium SB103 (media prepared the sameas for SB 166, except no activated charcoal is included) for 3 weeks.During this period, individual embryos can be removed from the clustersand screened for alterations in their fatty acid compositions. It shouldbe noted that any detectable phenotype, resulting from the expression ofthe genes of interest, could be screened at this stage. This wouldinclude, but not be limited to: alterations in fatty acid profile,protein profile and content, carbohydrate content, growth rate,viability or the ability to develop normally into a soybean plant.

Embryo Desiccation And Germination: Matured individual embryos will bedesiccated by placing them into an empty, small petri dish (35×10 mm)for approximately 4-7 days. The plates are sealed with fiber tape(creating a small humidity chamber). Desiccated embryos are planted intoSB71-4 medium where they are left to germinate under the same cultureconditions described above.

SB 714 Solid Medium (Per Liter)

1 bottle Gamborg's B5 salts w/ sucrose (Gibco/BRL, Catalog #21153-036)

pH 5.7

5 g TC agar

Germinated plantlets will be removed from germination medium and rinsedthoroughly with water and then planted in Redi-Earth in 24-cell packtrays, covered with clear plastic domes. After 2 wks, the domes will beremoved and plants hardened off for a further week. If plantlets lookhardy, they are transplanted to 10″ pots of Redi-Earth with up to 3plantlets per pot. After 10-16 wks, mature seeds will be harvested,chipped and analyzed for fatty acids.

Example 10 Analysis of Somatic Soy Embryos Comprising the Fusariummoniliforme Δ15 Desaturase of Sub-Family 1 (Fm1)

This Example describes methods that will be useful to analyze fatty acidcontent in transformant soybean comprising a chimeric gene containingthe Fm1 Δ15 desaturase coding region.

Theory

Mature somatic soybean embryos are a good model for zygotic embryos.While in the globular embryo state in liquid culture, somatic soybeanembryos contain very low amounts of triacylglycerol or storage proteinstypical of maturing, zygotic soybean embryos. At this developmentalstage, the ratio of total triacylglyceride to total polar lipid(phospholipids and glycolipid) is about 1:4, as is typical of zygoticsoybean embryos at the developmental stage from which the somatic embryoculture was initiated. At the globular stage as well, the mRNAs for theprominent seed proteins, α′-subunit of β-conglycinin, kunitz trypsininhibitor 3, and seed lectin are essentially absent. Upon transfer tohormone-free media to allow differentiation to the maturing somaticembryo state, triacylglycerol becomes the most abundant lipid class;and, mRNAs for α′-subunit of β-conglycinin, kunitz trypsin inhibitor 3and seed lectin become very abundant messages in the total mRNApopulation. On this basis, the somatic soybean embryo system behavesvery similarly to maturing zygotic soybean embryos in vivo, and istherefore a good and rapid model system for analyzing the phenotypiceffects of modifying the expression of genes in the fatty acidbiosynthetic pathway. Most importantly, the model system is alsopredictive of the fatty acid composition of seeds from plants derivedfrom transgenic embryos.

Fatty Acid Analysis

Transgenic somatic soybean embryos will be analyzed. For this, fattyacid methyl esters will be prepared from single, matured, somatic soyembryos by transesterification. Embryos will be placed in a vialcontaining 50 μL of trimethylsulfonium hydroxide (TMSH) and 0.5 mL ofhexane and incubated for 30 min at room temperature while shaking. Fattyacid methyl esters (5 μL injected from hexane layer) will be separatedand quantified using a Hewlett-Packard 6890 Gas Chromatograph fittedwith an Omegawax 320 fused silica capillary column (Supelco Inc.,Bellefonte, Pa.; Catalog #24152). A portion of the somatic embryos willhave higher levels of ALA than in control somatic embryos.

Mature plants will be regenerated from transformed embryos, and thefatty acid analyses will be performed on the seeds that are produced bythe regenerated plants. These plants will then be crossed with othertransgenic plants expressing ω-3 fatty acid biosynthetic pathway genes(wherein the combined levels of EPA and DPA are frequently greater than15%, and are as high as 23.5% of the total). Representative genespreferred for making long-chain PUFAs (e.g., EPA) include one or more ofthe following: TABLE 13 EPA Biosynthetic Pathway Genes Plasmid GeneOrganism Name Reference Δ6 desaturase S. diclina pRSP1 WO 02/081668 Δ6desaturase M. alpina pCGR5 U.S. Pat. No. 5,968,809 Elongase M. alpinapRPB2 WO 00/12720 Elongase T. aureum pRAT-4-A7 WO 02/08401 Δ5 desaturaseM. alpina pCGR4 U.S. Pat. No. 6,075,183 Δ5 desaturase S. diclina pRSP3WO 02/081668 Δ4 desaturase S. aggregatum pRSA1 WO 02/090493

Example 11 Cloning the Fusarium Δ15 Desaturase into a Soybean ExpressionVector (pKR578)

This example describes the construction of pKR578, a vector for strong,seed-specific expression of the Δ15 desaturase in soybeans.

Vector pKS121 (WO 02/00904) contains a Not[ site flanked by the Kunitzsoybean Trypsin Inhibitor (KTi) promoter [Jofuku et al., (1989) PlantCell 1:1079-1093] and the KTi 3′ termination region, the isolation ofwhich is described in U.S. Pat. No. 6,372,965 (KTi/NotI/KTi3′ cassette).Vector pKR457 is a derivative of pKS121 where the restriction sitesupstream and downstream of the Kti/NotI/Kti3′ cassette have been alteredthrough a number of subcloning steps. Vector pKR457 also contains theSoy albumin transcription terminator downstream of the Kti terminator tolengthen and strengthen termination of transcription. In pKR457, theBamHI site upstream of the Kti promoter in pKS121 was removed and a newsequence (SEQ ID NO:73) added containing a BsiWI, SalI, SbfI and HindIIIsite with the BsiWI site being closest the 5′ end of the Kti promoter.In addition, the SalI site downstream of the Kti terminator in pKS121was removed and a new sequence (SEQ ID NO: 74) added containing an XbaI(closest to 3′ end of Kti terminator), a BamHI site, the soy albumintranscription terminator sequence, a BsiWI site and another BamHI site(Kti/NotI/KtiSalb cassette). The albumin transcription terminator waspreviously amplified from soy genomic DNA using primer oSalb-12 (SEQ IDNO: 75), designed to introduce BamHI, XbaI and BsiWI sites at the 3′ endof the terminator, and primer oSalb-13 (SEQ ID NO: 76), designed tointroduce BamHI sites at the 5′ end of the terminator. .

A starting plasmid pKS123 (WO 02/08269, the contents of which are herebyincorporated by reference) contains the hygromycin B phosphotransferasegene (HPT) [Gritz, L. and Davies, J. (1983) Gene 25:179-188], flanked bythe T7 promoter and transcription terminator (T7prom/hpt/T7termcassette), and a bacterial origin of replication (ori) for selection andreplication in bacteria such as E. coli. In addition, pKS123 alsocontains the hygromycin B phosphotransferase gene, flanked by the 35Spromoter [Odell et al., (1985) Nature 313:810-812] and NOS 3′transcription terminator [Depicker et al., (1982) J. Mol. Appl. Genet1:561:570] (35S/hpt/NOS3′ cassette) for selection in plants such assoybean. pKS123 also contains a NofI restriction site, flanked by thepromoter for the α′ subunit of β-conglycinin [Beachy et al., (1985) EMBOJ. 4:3047-3053] and the 3′ transcription termination region of thephaseolin gene [Doyle, J. J. et al. (1986) J. Biol. Chem. 261:9228-9238]thus allowing for strong tissue-specific expression in the seeds ofsoybean of genes cloned into the NotI site. Vector pKR72 is a derivativepKS123 where the HindIII fragment containing theβ-conglycinin/NotI/phaseolin cassette has been inverted and a sequence(SEQ ID NO:77) containing SbfI, FseI and BsiWI restriction enzyme siteswas introduced between the HindIII and BamHI sites in front of theβ-conglycinin promoter. Vector pKR72 was digested with HindIII to removethe Pcon/NotI/Phas3′ cassette and give pKR325.

An intermediate cloning vector was formed by cloning the BsiWI fragmentof pKR457, containing the Kti/NotI/KtiSalb cassette into the BsiWI siteof pKR325. The NodI fragment of pY34 (see Example 4) containing theFusarium Δ15 desaturase was then cloned into the NotI site of thisintermediate vector to give pKR578. Plasmid pKR578 (SEQ ID NO:78) isshown in FIG. 6. Plasmid pKR578 has been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, bearing ATCC accession number PTA-XXXX with a date ofdeposit of Nov. 4, 2004.

Example 12 Isolation of Soybean Seed-Specific Promoters

The cloning of soybean seed-specific promoters has been described in WO04/071467 and is re-described here.

The soybean annexin and BD30 promoters (described in WO 04/071178,published on Aug. 26, 2004) were isolated with the UniversalGenomeWalker system (Clontech) according to its user manual (PT3042-1).To make soybean GenomeWalker libraries, samples of soybean genomic DNAwere digested with DraI, EcoRV, PvuII and StuI separately for two hours.After DNA purification, the digested genomic DNAs were ligated to theGenomeWalker adaptors AP1 and AP2.

Two gene specific primers (GSP1 and GSP2) were designed for soybeanannexin gene based on the 5′ coding sequences in annexin cDNA in DuPontEST database. The sequences of GSP1 and GSP2 are set forth in SEQ IDNOS:79 and 80.

The AP1 and the GSP1 primers were used in the first round PCR using theconditions defined in the GenomeWalker system protocol. Cycle conditionswere 94° C. for 4 minutes; 94° C. for 2 second and 72° C. for 3 minutes,7 cycles; 94° C. for 2 second and 67° C. for 3 minutes, 32 cycles; 67°C. for 4 minutes. The products from the first run PCR were diluted50-fold. One microliter of the diluted products were used as templatesfor the second PCR with the AP2 and GSP2 as primers. Cycle conditionswere 94° C. for 4 minutes; 94° C. for 2 second and 72° C. for 3 min, 5cycles; 94° C. for 2 second and 67° C. for 3 minutes, 20 cycles; 67° C.for 3 minutes. A 2.1 kb genomic fragment was amplified and isolated fromthe EcoRV-digested GenomeWalker library. The genomic fragment wasdigested with BamH I and Sal I and cloned into Bluescript KS+vector forsequencing. The DNA sequence of this 2012 bp soybean annexin promoterfragment is set forth in SEQ ID NO:81.

Two gene specific primers (GSP3 and GSP4) were designed for soybean BD30based on the 5′ coding sequences in BD30 cDNA in NCBI GenBank (J05560).The oligonucleotide sequences of the GSP3 and GSP4 primers have thesequences set forth in SEQ ID NOS:82 and 83.

The AP1 and the GSP3 primers were used in the first round PCR using thesame conditions defined in the GenomeWalker system protocol. The cycleconditions used for soybean annexin promoter do not work well for thesoybean BD30 promoter in GenomeWalker experiment. A modified touchdownPCR protocol was used. Cycle conditions were: 94° C. for 4 minutes; 94°C. for 2 second and 74° C. for 3 minutes, 6 cycles in which annealingtemperature drops 1° C. every cycle; 94° C. for 2 second and 69° C. for3 minutes, 32 cycles; 69° C. for 4 minutes. The products from the 1^(st)run PCR were diluted 50-fold. One microliter of the diluted productswere used as templates for the 2^(nd) PCR with the AP2 and GSP4 asprimers. Cycle conditions were: 94° C. for 4 minutes; 94° C. for 2second and 74° C. for 3 min, 6 cycles in which annealing temperaturedrops 1° C. every cycle; 94° C. for 2 second and 69° C. for 3 minutes,20 cycles; 69° C. for 3 minutes. A 1.5 kb genomic fragment was amplifiedand isolated from the PvuII-digested GenomeWalker library. The genomicfragment was digested with BamHI and SalI and cloned into Bluescript KS⁺vector for sequencing. DNA sequencing determined that this genomicfragment contained a 1408 bp soybean BD30 promoter sequence (SEQ IDNO:84).

Based on the sequences of the soybean β-conglycinin β-subunit promotersequence in NCBI database (S44893), two oligos with either BamHI or NotIsites at the 5′ ends were designed to amplify the soybean β-conglycininβ-subunit promoter (SEQ ID NO:85). The oligonucleotide sequences ofthese two oligos are set forth in SEQ ID NOS: 86 and 87.

Based on the sequences of the soybean Glycinin Gy1 promoter sequence inthe NCBI GenBank database (X15121), two oligos with either BamHI or NotIsites at the 5′ ends were designed to amplify the soybean Glycinin Gy1promoter (SEQ ID NO:88). The oligonucleotide sequences of these twooligos are set forth in SEQ ID NOS:89 and 90.

Example 13 Cloning the Fusarium Δ15 Desaturase into a Soybean ExpressionVector for Co-expression with a Δ17 Desaturase (PKR585)

This example describes the construction of pKR585, a vector for strong,seed-specific expression of the Fusarium Δ15 desaturase and Saprolegniadiclina Δ17 desaturase in soybeans. Construction of an intermediatecloning vector (pKR271), containing the Saprolegnia diclina Δ17desaturase [Pereira et al. (2004) Biochem. J. 378, 665-671] undercontrol of the soy annexin promoter, has previously been described in WO04/071467 and is re-stated here.

The KTi/NotI/KTi3′ cassette was PCR-amplified from pKS121 using primersoKTi5 (SEQ ID NO:91) and oKTi6 (SEQ ID NO:92), designed to introduce anXbaI and BsiWI site at both ends of the cassette. The resulting PCRfragment was subcloned into the XbaI site of the cloning vector pUC19 togive plasmid pKR124 thus adding a PstI and SbfI site at the 3′ end ofthe Kti transcription terminator.

The SalI fragment of pJS93 containing soy BD30 promoter (WO 01/68887)was combined with the SalI fragment of pUC19 to give pKR227 thus addinga PstI and SbfI site at the 5′ end of the BD30 promoter.

The BD30 3′ transcription terminator was PCR-amplified from soy genomicDNA using primer oSBD30-1 (SEQ ID NO:93), designed to introduce an NotIsite at the 5′ end of the terminator, and primer oSBD30-2 (SEQ IDNO:94), designed to introduce a BsiWI site at the 3′ end of theterminator.

The resulting PCR fragment was subcloned into the intermediate cloningvector pCR-Script AMP SK(+) (Stratagene) according the manufacturer'sprotocol to give plasmid pKR251r. The EcoRI/NotI fragment from pKR251r,containing the BD30 3′ transcription terminator, was cloned into theEcoRI/NotI fragment of intermediate cloning vector pKR227 to givepKR256.

The annexin promoter (SEQ ID NO:81) from pJS92 was released by BamHIdigestion and the ends were filled. The resulting fragment was ligatedinto the filled BsiWI fragment from the vector backbone of pKR124 in adirection which added a PsfI and SbfI site at the 5′ end of the annexinpromoter to give pKR265. The annexin promoter was released from pKR265by digestion with SbfI and NotI and was cloned into the SbfI/NotIfragment of pKR256, containing the BD30 3′ transcription terminator, anampicillin resistance gene and a bacterial ori region, to give pKR268.

The gene for the Saprolegnia diclina Δ17 desaturase was released frompKS203 [Pereira et al. (2004) Biochem. J. 378, 665-671] by partialdigestion with NotI, and was cloned into the NotI site of pKR268 to givepKR271.

Plasmid pKR271 was then digested with PstI and the fragment containingthe Saprolegnia diclina Δ17 desaturase was cloned into the SbfI site ofpKR578 to give pKR585. In this way, the Fusarium Δ15 desaturase could beco-expressed with the Saprolegnia diclina Δ17 desaturase behind strong,seed-specific promoters. A map of pKR585 (SEQ ID NO:95) is shown in FIG.7. Plasmid pKR585 has been deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209,bearing ATCC accession number PTA-XXXX with a date of deposit of Nov. 4,2004.

Example 14 Assembling EPA biosynthetic Pathway Genes for Expression inSoybeans (pKR274)

This example describes the construction of pKR274, a vector designed forstrong, seed-specific expression of the M. alpina Δ6 desaturase (U.S.Pat. No. 5,968,809), M. alpina elongase (WO 00/12720) and M. alpina Δ5desaturase (U.S. Pat. No. 6,075,183) in somatic soybean embryos andsoybean seeds. Construction of this vector was previously described inWO 04/071467 and is re-stated here.

The Δ6 desaturase was cloned behind the promoter for the α′ subunit ofβ-conglycinin [Beachy et al., (1985) EMBO J. 4:3047-3053] followed bythe 3′ transcription termination region of the phaseolin gene [Doyle, J.J. et al. (1986) J. Biol. Chem. 261:9228-9238] (βcon/Mad6/Phas3′cassette).

The Δ5 desaturase was cloned behind the Kunitz soybean Trypsin Inhibitor(KTi) promoter [Jofuku et al., (1989) Plant Cell 1:1079-1093], followedby the KTi 3′ termination region, the isolation of which is described inU.S. Pat. No.6,372,965 (KTi/Mad5/KTi3′ cassette).

The elongase was cloned behind the glycinin Gy1 promoter (SEQ ID NO:88)followed by the pea leguminA2 3′ termination region (Gy1/Maelo/legA2cassette).

All of these promoters exhibit strong tissue specific expression in theseeds of soybean. Plasmid pKR274 also contains the hygromycin Bphosphotransferase gene [Gritz, L. and Davies, J. (1983) Gene25:179-188] cloned behind the T7 RNA polymerase promoter and followed bythe T7 terminator (T7prom/HPT/T7term cassette) for selection of theplasmid on hygromycin B in certain strains of E. coli, such asNovaBlue(DE3) (Novagen, Madison, Wis.), which is lysogenic for lambdaDE3 (and carries the T7 RNA polymerase gene under lacUV5 control). Inaddition, plasmid pKR274 contains a bacterial origin of replication (on)functional in E. coli from the vector pSP72 (Stratagene).

The gene for the M. alpina Δ6 desaturase was PCR-amplified from pCGR5(U.S. Pat. No. 5,968,809) using primers oCGR5-1 (SEQ ID NO:96) andoCGR5-2 (SEQ ID NO:97), which were designed to introduce NotIrestriction enzyme sites at both ends of the Δ6 desaturase and an NcoIsite at the start codon of the reading frame for the enzyme.

The resulting PCR fragment was subcloned into the intermediate cloningvector pCR-Script AMP SK(+) (Stratagene) according the manufacturer'sprotocol to give plasmid pKR159.

The NotI fragment of pKR159, containing the M. alpina Δ6 desaturasegene, was cloned into NofI site of pZBL117 in the sense orientation tomake plant expression cassette pZBL119.

Vector pKR197 was constructed by combining the AscI fragment fromplasmid pKS102 (WO 02/00904), containing the T7prom/hpt/T7term cassetteand bacterial ori, with the AscI fragment of plasmid pKR72, containingthe βcon/NotI/Phas cassette.

Plasmid pKR159 was digested with NotI to release the M. alpina Δ6desaturase, which was, in turn, cloned into the NotI site of the soybeanexpression vector pKR197 to give pKR269.

The glycininGy1 promoter was amplified from pZBL119 using primer oSGly-1(SEQ ID NO:98), designed to introduce an SbfI/PstI site at the 5′ end ofthe promoter, and primer oSGly-2 (SEQ ID NO:99), designed to introduce aNotI site at the 3′ end of the promoter.

The resulting PCR fragment was subcloned into the intermediate cloningvector pCR-Script AMP SK(+) (Stratagene) according the manufacturer'sprotocol to give plasmid pSGly12.

The legA2 promoter was amplified from pea genomic DNA using primerLegPro5′ (SEQ ID NO:100), designed to introduce XbaI and BsiWI sites atthe 5′ end of the promoter, and primer LegPro3′ (SEQ ID NO:101),designed to introduce a NotI site at the 3′ end of the promoter.

The legA2 transcription terminator was amplified from pea genomic DNAusing primer LegTerm5′ (SEQ ID NO:102), designed to introduce NotI siteat the 5′ end of the terminator, and primer LegTerm3′ (SEQ ID NO:103),designed to introduce BsiWI and XbaI sites at the 3′ end of theterminator.

The resulting PCR fragments were then combined and re-amplified usingprimers LegPro5′ and LegTerm3′, thus forming the legA2/NotI/legA23′cassette. The legA2/NotI/legA23′ cassette PCR fragment was subclonedinto the intermediate cloning vector pCR-Script AMP SK(+) (Stratagene)according the manufacturer's protocol to give plasmid pKR140. PlasmidpKR142 was constructed by cloning the BsiWI fragment of pKR140,containing the legA2/NotI/legA23′ cassette, into the BsNVI site ofpKR124, containing a bacterial ori and ampicillin resistance gene. ThePstI/NotI fragment from plasmid pKR142 was then combined with thePstI/NotI fragment of plasmid pSGly12, containing the glycininGy1promoter, to give pKR263.

The gene for the M. alpina Δ5 desaturase was amplified from pCGR4 (U.S.Pat. No. 6,075,183) using primers CGR4forward (SEQ ID NO:104) andCGR4reverse (SEQ ID NO:105) which were designed to introduce NotIrestriction enzyme sites at both ends of the desaturase.

The resulting PCR fragment was digested with NotI and cloned into theNotI site of vector pKR124 to give pKR136.

The gene for the Mortierella alpina elongase was amplified from pRPB2(WO 00/12720) using primers RPB2foward (SEQ ID NO:106) and RPB2reverse(SEQ ID NO:107) which were designed to introduce NotI restriction enzymesites at both ends of the elongase. The resulting PCR fragment wasdigested with NotI and cloned into the NotI site of vector pKR263 togive pKR270.

The Gy1/Maelo/legA2 cassette was released from plasmid pKR270 bydigestion with BsiWI and SbfI and was cloned into the BsiWI/SbfI sitesof plasmid pKR269, containing the Δ6 desaturase, the T7prom/hpt/T7termcassette and the bacterial ori region. This was designated as plasmidpKR272.

The KTi/Mad5/KTi3′ cassette, released from pKR1 36 by digestion withBsiWI, was then cloned into the BsiWI site of pKR272 to give pKR274(FIG. 8).

Example 15 Assembling EPA Biosynthetic Pathway Genes for Expression inSoybeans (pKKE2)

This example describes the construction of pKKE2, a vector designed forstrong, seed-specific expression of the Saprolegnia diclina Δ6desaturase (WO 02/081668), M. alpina elongase (WO 00/12720) and M.alpina 66 5 desaturase (U.S. Pat. No. 6,075,183) in somatic soybeanembryos and soybean seeds. This vector is identical to pKR274 exceptthat the M. alpina Δ6 desaturase has been replaced with the theSaprolegnia diclina Δ6 desaturase. Construction of this vector waspreviously described in WO 04/071467 and is re-stated here.

The S. diclina Δ6 desaturase was removed from pRSP1 (WO 02/081668) bydigestion with EcoRI and HindIII. The ends of the resulting DNA fragmentwere filled and the fragment was cloned into the filled NotI site ofpKS123 to give pKS208.

The βcon/Sdd6/Phas3′ cassette was released from plasmid pKS208 bydigestion with HindIII and was cloned into the HindIII site of plasmidpKR272 to give pKR301.

The KTi/Mad5/KTi3′ cassette, released from pKR136, by digestion withBsiWI, was then cloned into the BsiWI site of pKR301 to give pKKE2 (FIG.9).

Example 16

Transformation of Somatic Soybean Embryo Cultures Culture Conditions

Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35ml liquid medium SB196 (see recipes below) on a rotary shaker, 150 rpm,26° C. with cool white fluorescent lights on 16:8 hr day/nightphotoperiod at light intensity of 60-85 μE/m2/s. Cultures aresubcultured every 7 days to two weeks by inoculating approximately 35 mgof tissue into 35 ml of fresh liquid SB196 (the preferred subcultureinterval is every 7 days).

Soybean embryogenic suspension cultures are transformed with pKR353(Δ15) plasmid described in the Example 18 by the method of particle gunbombardment (Klein et al. 1987; Nature, 327:70). A DuPont BiolisticPDS1000/HE instrument (helium retrofit) is used for all transformations.

Soybean Embryogenic Suspension Culture Initiation

Soybean cultures are initiated twice each month with 5-7 days betweeneach initiation.

Pods with immature seeds from available soybean plants 45-55 days afterplanting are picked, the seeds removed from their shells and placed intoa sterilized magenta box. The soybean seeds are sterilized by shakingthem for 15 minutes in a 5% Clorox solution with 1 drop of ivory soap(95 ml of autoclaved distilled water plus 5 ml Clorox and 1 drop ofsoap). Mix well. Seeds are rinsed using 2 1-liter bottles of steriledistilled water and those less than 4 mm are placed on individualmicroscope slides. The small end of the seed is cut and the cotyledonspressed out of the seed coat. Cotyledons are transferred to platescontaining SB1 medium (25-30 cotyledons per plate). Plates are wrappedwith fiber tape and stored for 8 weeks. After this time secondaryembryos are cut and placed into SB196 liquid media for 7 days.

Preparation of DNA for Bombardment

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene is used for bombardment.Fragments are obtained by gel isolation of double digested plasmids. Ineach case, 100 ug of plasmid DNA is digested in 0.5 ml of theappropriate enzyme mix. The resulting DNA fragments are separated by gelelectrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker MolecularApplications) and the DNA fragments containing chimeric genes are cutfrom the agarose gel. DNA is purified from the agarose using the GELasedigesting enzyme following the manufacturer's protocol.

A 50 μl aliquot of sterile distilled water containing 3 mg of goldparticles (3 mg gold) is added to 5 μl of a 1 μg/μl DNA solution (eitherintact plasmid or DNA fragment prepared as described above), 50 μl 2.5MCaCl₂ and 20 μl of 0.1 M spermidine. The mixture is shaken 3 min onlevel 3 of a vortex shaker and spun for 10 sec in a bench microfuge.After a wash with 400 μl 100% ethanol the pellet is suspended bysonication in 40 μl of 100% ethanol. Five μl of DNA suspension isdispensed to each flying disk of the Biolistic PDS1000/HE instrumentdisk. Each 5 μl aliquot contains approximately 0.375 mg gold perbombardment (i.e. per disk).

Tissue Preparation and Bombardment with DNA

Approximately 150-200 mg of 7 day old embryonic suspension cultures areplaced in an empty, sterile 60×15 mm petri dish and the dish coveredwith plastic mesh. Tissue is bombarded 1 or 2 shots per plate withmembrane rupture pressure set at 1100 PSI and the chamber evacuated to avacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5inches from the retaining/stopping screen.

Selection of Transformed Embryos

Transformed embryos are selected either using hygromycin (when thehygromycin phosphotransferase, HPT, gene is used as the selectablemarker) or chlorsulfuron (when the acetolactate synthase, ALS, gene isused as the selectable marker).

Hygromycin (HPT) Selection

Following bombardment, the tissue is placed into fresh SB196 media andcultured as described above. Six days post-bombardment, the SB196 isexchanged with fresh SB196 containing a selection agent of 30 mg/Lhygromycin. The selection media is refreshed weekly. Four to six weekspost selection, green, transformed tissue may be observed growing fromuntransformed, necrotic embryogenic clusters. Isolated, green tissue isremoved and inoculated into multiwell plates to generate new, clonallypropagated, transformed embryogenic suspension cultures.

Chlorsulfuron (ALS) Selection

Following bombardment, the tissue is divided between 2 flasks with freshSB196 media and cultured as described above. Six to seven dayspost-bombardment, the SB196 is exchanged with fresh SB196 containingselection agent of 0.1 mg/L Chlorsulfuron. The selection media isrefreshed weekly. Four to six weeks post selection, green, transformedtissue may be observed growing from untransformed, necrotic embryogenicclusters. Isolated, green tissue is removed and inoculated intomultiwell plates containing SB196 to generate new, clonally propagated,transformed embryogenic suspension cultures.

Regeneration of Soybean Somatic Embryos into Plants

In order to obtain whole plants from embryogenic suspension cultures,the tissue must be regenerated.

Embryo Maturation

Embryos are cultured for 4-6 weeks at 26° C. in SB1 96 under cool whitefluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro(Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with lightintensity of 90120 uE/m2s. After this time embryo clusters are removedto a solid agar media, SB166, for 1-2 weeks. Clusters are thensubcultured to medium SB103 for 3 weeks. During this period, individualembryos can be removed from the clusters and screened for alterations intheir fatty acid compositions. It should be noted that any detectablephenotype, resulting from the expression of the genes of interest, couldbe screened at this stage. This would include, but not be limited to,alterations in fatty acid profile, protein profile andcontent,.carbohydrate content, growth rate, viability, or the ability todevelop normally into a soybean plant.

Embryo Desiccation and Germination

Matured individual embryos are desiccated by placing them into an empty,small petri dish (35×10 mm) for approximately 4-7 days. The plates aresealed with fiber tape (creating a small humidity chamber). Desiccatedembryos are planted into SB71-4 medium where they are left to germinateunder the same culture conditions described above. Germinated plantletsare removed from germination medium and rinsed thoroughly with water andthen planted in Redi-Earth in 24-cell pack trays, covered with clearplastic domes. After 2 weeks the domes are removed and plants hardenedoff for a further week. If plantlets look hardy they are transplanted to10″ pot of Redi-Earth with up to 3 plantlets per pot. After 10 to 16weeks, mature seeds are harvested, chipped and analyzed for fatty acids.Media Recipes SB 196 - FN Lite liquid proliferation medium (per liter) -MS FeEDTA - 100× Stock 1 10 ml MS Sulfate - 100× Stock 2 10 ml FN LiteHalides - 100× Stock 3 10 ml FN Lite P, B, Mo - 100× Stock 4 10 ml B5vitamins (1 ml/L) 1.0 ml 2,4-D (10 mg/L final concentration) 1.0 ml KNO32.83 gm (NH4)2 SO 4 0.463 gm Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8FN Lite Stock Solutions Stock # 1000 ml 500 ml 1 MS Fe EDTA 100× StockNa₂ EDTA* 3.724 g 1.862 g FeSO₄—7H₂O 2.784 g 1.392 g 2 MS Sulfate 100×stock MgSO₄—7H₂O 37.0 g 18.5 g MnSO₄—H₂O 1.69 g 0.845 g ZnSO₄—7H₂O 0.86g 0.43 g CuSO₄—5H₂O 0.0025 g 0.00125 g 3 FN Lite Halides 100× StockCaCl₂—2H₂O 30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl₂—6H₂O 0.0025 g 0.00125g 4 FN Lite P, B, Mo 100× Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 gNa₂MoO₄—2H₂O 0.025 g 0.0125 g*Add first, dissolve in dark bottle while stirring

SB1 Solid Medium (Per Liter)

b 1 pkg. MS salts (Gibco/ BRL—Cat# 11117-066)

1 ml B5 vitamins 1000× stock

31.5 g sucrose

2 ml 2,4-D (20mg/L final concentration)

pH 5.7

8 g TC agar

SB 166 Solid Medium (Per Liter)

1 pkg. MS salts (Gibco/ BRL—Cat# 11117-066)

1 ml B5 vitamins 1000× stock

60 g maltose

750 mg MgCl2 hexahydrate

5 g activated charcoal

pH 5.7

2 g geirite

SB 103 Solid Medium (Per Liter)

1 pkg. MS salts (GibcoIBRL—Cat# 11 117-066)

1 ml B5 vitamins 1000× stock

60 g maltose

750 mg MgCl2 hexahydrate

pH 5.7

2 g gelrite

SB 71-4 Solid Medium (Per Liter)

1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL—Cat# 21153-036)

pH 5.7

5 g TC agar

2,4-D Stock

obtained premade from Phytotech cat# D 295—concentration is 1 mg/ml

B5 Vitamins Stock (Per 100 ml)—Store Aliquots at −20C

10 g myo-inositol

100 mg nicotinic acid

100 mg pyridoxine HCl

1 g thiamine

If the solution does not dissolve quickly enough, apply a low level ofheat via the hot stir plate.

Chlorsulfuron Stock

1mg / ml in 0.01 N Ammonium Hydroxide

Example 17 Analysis of Somatic Soy Embryos containing the Fusarium Δ15Desaturase

Mature somatic soybean embryos are a good model for zygotic embryos.While in the globular embryo state in liquid culture, somatic soybeanembryos contain very low amounts of triacylglycerol or storage proteinstypical of maturing, zygotic soybean embryos. At this developmentalstage, the ratio of total triacylglyceride to total polar lipid(phospholipids and glycolipid) is about 1:4, as is typical of zygoticsoybean embryos at the developmental stage from which the somatic embryoculture was initiated. At the globular stage as well, the mRNAs for theprominent seed proteins, α′-subunit of β-conglycinin, kunitz trypsininhibitor 3, and seed lectin are essentially absent. Upon transfer tohormone-free media to allow differentiation to the maturing somaticembryo state, triacylglycerol becomes the most abundant lipid class. Aswell, mRNAs for α′-subunit of β-conglycinin, kunitz trypsin inhibitor 3and seed lectin become very abundant messages in the total mRNApopulation. On this basis somatic soybean embryo system behaves verysimilarly to maturing zygotic soybean embryos in vivo, and is thereforea good and rapid model system for analyzing the phenotypic effects ofmodifying the expression of genes in the fatty acid biosynthesis pathway(Example 3 in WO 02/00904). Most importantly, the model system is alsopredictive of the fatty acid composition of seeds from plants derivedfrom transgenic embryos.

Transgenic somatic soybean embryos containing the constructs describedabove were analyzed in a similar way. For this, fatty acid methyl estersare prepared from single, matured, somatic soy embryos bytransesterification. Embryos are placed in a vial containing 50 μL oftrimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane and incubatedfor 30 minutes at room temperature while shaking. Fatty acid methylesters (5 μL injected from hexane layer) are separated and quantifiedusing a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax320 fused silica capillary column (Supelco Inc., Cat#24152). The oventemperature was programmed to hold at 220° C. for 2.7 min, increase to240° C. at 20° C. /min and then hold for an additional 2.3 min. Carriergas was supplied by a Whatman hydrogen generator. Retention times werecompared to those for methyl esters of standards commercially available(Nu-Chek Prep, Inc. catalog #U-99-A).

Results for the preferred 10 lines containing pKR578 as well as thosefor a control embryo transformed with selection only are shown in Table14. Although lines for only the preferred embryos are shown, other lineshaving ALA levels ranging from the control (22%) up to the highest (89%)were obtained. Similarly, others lines having omega-3 to omega-6 ratiosranging from 0.4 to 45 were obtained. The preferred line had embryoswith an average 18:3 content of 79% with the highest embryo analyzedhaving 89% 18:3, versus the control which had an average 18:3 content of19% and a highest embryo of 22% 18:3. This corresponds to an average4-fold improvement in 18:3 when compared to control embryos. The 18:3content range in the lines transformed with pKR578 is 51-89%. This linealso had an average ratio of omega-3:omega-6 fatty acids (18:3/18:2) of24:1 with the highest embryo having a ratio of 42:1, versus the controlwhich had an average and highest 18:3/18:2 ratio of 0.4. Thiscorresponds to an average 66-fold improvement in omega-3:omega-6 ratios.The ratio range of omega-3:omega-6 fatty acids (18:3/18:2) in the linestransformed with pKR578 was 3:142:1. TABLE 14 Accumulation of 18:3 (ALA)in lines transformed with pKR578 18:3 18:3 18:3/ Ave High Line* 16:018:0 18:1 18:2 18:3 (ave) high 18:2 ratio ratio Control 1566: 5-11-1 172 7 52 22 0.4 5-11-2 17 2 9 53 19 0.4 5-11-3 15 3 10 57 14 19 22 0.2 0.40.4 5-11-4 17 3 8 53 18 0.3 5-11-5 16 4 16 44 19 0.4 +pKR578 1566:5-15-1 11 2 10 6 70 12 5-15-2 16 2 9 8 65 8 5-15-3 16 3 13 9 59 62 70 78 12 5-15-4 17 4 17 12 51 4 5-15-5 15 2 10 8 65 8 +pKR578 1566: 8-5-1 142 10 14 59 4 8-5-2 13 3 9 7 68 10 8-5-3 14 3 10 6 67 63 68 11 7 10 8-5-417 3 10 11 59 5 8-5-5 14 2 12 12 59 5 +pKR578 1566: 7-6-1 19 3 12 5 6212 7-6-2 15 2 8 8 67 8 7-6-3 20 5 13 3 59 63 74 18 14 27 7-6-4 16 2 1411 56 5 7-6-5 17 2 4 3 74 27 +pKR578 1573: 9-4-1 14 3 11 8 64 8 9-4-2 152 11 7 64 9 9-4-3 16 2 14 15 53 61 65 4 7 10 9-4-4 11 3 13 14 59 4 9-4-517 3 10 6 65 10 +pKR578 1573: 10-4-1 18 3 6 4 70 19 10-4-2 16 4 9 2 6967 70 29 18 29 10-4-3 16 2 11 9 62 7 10-4-4 17 2 10 4 66 17 +pKR5781582: 2-2-1 0 2 9 8 81 10 2-2-2 15 2 11 5 67 13 2-2-3 0 1 8 2 89 79 8942 24 42 2-2-4 0 1 7 3 89 31 2-2-5 12 1 7 3 77 24 2-2-6 14 1 7 5 73 15+pKR578 1582: 2-3-1 17 3 10 10 60 6 2-3-2 16 2 9 9 65 7 2-3-3 16 2 8 1362 61 65 5 5 7 2-3-4 17 2 8 17 56 3 2-3-5 17 2 11 12 58 5 2-3-6 16 2 9 964 7 +pKR578 1582: 2-6-1 16 2 8 8 67 8 2-6-2 17 2 7 6 68 11 2-6-3 17 2 76 68 66 69 11 8 11 2-6-4 16 2 8 12 61 5 2-6-5 17 2 9 12 61 5 2-6-6 16 26 7 69 10 +pKR578 1582: 3-1-1 17 2 15 8 58 7 3-1-2 15 2 8 10 65 7 3-1-318 2 5 2 73 66 73 45 15 45 3-1-4 18 2 7 7 66 9 3-1-5 16 2 7 7 68 103-1-6 16 2 10 4 69 20 +pKR578 1566: 7-5-1 16 2 7 2 73 36 7-5-2 6 2 6 483 23 7-5-3 14 2 9 8 67 72 83 9 19 36 7-5-4 15 2 8 7 68 10 7-5-5 15 2 75 71 15 7-5-6 14 2 8 7 69 9

Results for the preferred line containing pKKE2 and pKR585 are shown inTable 15. The preferred line had embryos with an average omega-3 contentof 63% and an average EPA content of 7%. The highest omega-3 embryoanalyzed had an omega-3 content of 72%. The highest EPA embryo analyzedhad EPA at 16% with the omega-3 content at 57%. This line also had anaverage ratio of omega-3:omega-6 fatty acids (18:3/18:2) of 8:1 with thehighest embryo having a ratio of 16:1. The highest EPA embryo had anomega-3:omega-6 ratio of 4:1. TABLE 15 Accumulation of omega-3 fattyacids in lines transformed with pKKE2 and pKR585 (Line 1491-15-2) TotalTotal ω3/ω Line 16:0 18:0 18:1 18:2 GLA 18:3 STA DGLA ARA ETA EPA DPAOther ω3 ω 6 6 1 14 3 4 7 1 59 3 1 0 1 6 0 3 71 8 9 2 18 4 7 11 8 24 8 21 3 12 0 2 48 22 2 3 15 2 4 4 1 62 3 1 0 2 4 0 2 72 6 11 4 16 3 3 6 0 584 0 0 2 6 0 2 71 7 10 5 15 3 6 6 1 57 2 1 0 2 5 0 2 68 9 8 6 15 2 4 7 439 9 2 0 4 12 0 2 65 13 5 7 20 6 8 11 7 18 7 3 1 4 14 1 3 44 22 2 8 18 57 4 6 17 14 3 0 7 16 2 1 57 13 4 9 20 5 3 9 2 36 5 2 1 4 10 1 3 58 15 410 16 3 6 4 0 63 1 0 0 0 1 0 4 70 5 16 11 17 4 6 5 0 61 2 0 0 0 2 0 3 685 13 12 18 5 9 4 0 56 1 0 0 0 1 0 6 64 4 16 13 15 5 8 5 0 57 1 0 0 1 4 14 67 5 13 14 20 7 3 7 1 42 4 1 0 2 7 1 5 61 9 7 15 17 3 6 5 1 55 3 1 0 16 0 2 68 6 11 16 18 4 5 9 2 47 3 1 0 2 7 1 3 61 12 5 Ave.: 17 4 6 6 2 474 1 0 2 7 1 3 63 10 8

Example 18 Transformation of Arabidopsis Plants

Vector pKR197 was digested with HindIII to remove the beta-conglycininexpression cassette and the vector backbone was re-ligated to givepKR277.

The Kti/NotI/Kti3′ cassette from pKR124 was removed by digestion withBsiWI, the ends filled in and the fragment cloned into the filledHindIII site of pKR277 to give pKR353.

The NotI fragment of pY34 containing the Fusarium Δ15 desaturase wascloned into the NotI site of pKR353 to give pKR353 (Δ15).

Vector pHD1 was derived from binary vector pZBL11 [U.S. Pat. No.5,968,793; EP 1003891; and WO 9859062] by adding an AscI linker betweenthe PacI and Asp718 sites between the right and left T-DNA borders. TheAscI linker was formed by annealing oligonucleotide Asc5 (SEQ ID NO:108)with Asc3 (SEQ ID NO:109).

Vector pZBL11 [U.S. Pat. No. 5,968,793; EP 1003891; and WO 9859062]contains a 35S:sulfonylurea resistant acetolactate synthase (ALS)transgene within the T-DNA borders that confers resistance tosulfonylurea herbicide and serves as the plant selectable marker. pZBL11also has an origin of replication for both E. coli and Agrobacteriumtumefaciens, and a bacterial kanamycin resistance gene.

The chimeric gene Kti3 promoter:Fm Δ15 desaturase ORF:Kti3 terminatorwas isolated as an Asc1 fragment from pKR353 (Δ15) and cloned into theunique Asc1 site in the binary vector pHD1 to give pZBLI(D15).

Plasmid PZBLI(D15) was transformed into Agrobacterium strain NTL4 [Luoet. al. (2001) MPMI 14:98] and this culture was used to transform afad2-1 mutant [Okuley et. al. (1994) Plant Cell 6: 147] of Arabidopsisthaliana by the Agrobacterium dip method. Transformants, given thedesignation NY, were selected on sulfonylurea, plants were grown and T2seed was obtained. Transformation was also carried out using pHD1 ascontrol in similar way. Lipid from bulk T2 seed batches (stillsegregating for the TDNA and sulfonylurea resistance) was analyzed asfollows. Approximately 25-50 T2 seeds were broken in 50 uL of TMSH usinga glass rod. After incubation at room temperature for approximately 15minutes with constant agitation, 500 uL of hexane was added the samplesincubated for an additional 15 minutes at room temperature withagitation. The hexane layer was then transferred to a separate GC vialand fatty acid methyl ester (FAME) analysis was carried out by GC asdescribed (WO 04/071467). Results for multiple lines are shown in Table16. The average 18:3 levels were approximately 1.5-fold higher in theΔ15-expressing lines (FmD15-NY) than in the empty vector control (HD1control) lines, while the 18:3/18:2 ratios were 2-fold higher in thesame lines. The n3/n6 ratio in wild type Arabidopsis is 0.61 [Shah et.al. (1997) Plant Physiology 114: 1533]. One skilled in the art wouldappreciate that the levels of ALA were underestimated because bulk seedwas analyzed that contained segregating seed (includes wild-type,hemizygous and homozygous seed). One skilled in the art would alsoappreciate that a homozygous lines would contain two times more copiesof Δ15 desaturase and thus, is expected to have higher levels of ALAthan heterzygous lines (gene dosage effect). TABLE 16 Accumulation of18:3 in a Fad2-1 mutant Arabidopsis transformed with the Fusarium Δ15Desaturase % % % % % % % ω3/ω 6 Sample 16:0 18:0 18:1 18:2 18:3 20:020:1 ratio HD1 control-1 5.2 2.2 62.4 2.4 7.3 0.8 19.6 3.1 HD1 control-25.3 2.2 63.1 2.2 7.1 0.9 19.3 3.3 HD1 control-3 6.1 2.5 59.2 2.8 8.9 0.919.6 3.2 HD1 control-4 5.5 2.3 60.9 2.3 8.0 1.0 20.0 3.4 HD1 control-55.5 2.1 61.6 2.9 7.6 0.8 19.6 2.7 HD1 control-6 5.4 2.3 61.8 2.1 7.2 0.920.2 3.4 HD1 control-7 5.3 2.6 61.8 2.3 7.6 1.0 19.3 3.2 HD1 control-85.2 2.0 63.0 2.9 7.5 0.8 18.6 2.6 HD1 control-9 5.2 2.2 62.9 2.3 8.5 0.818.1 3.6 HD1 control-10 5.7 2.3 61.1 2.5 8.3 0.9 19.3 3.3 HD1 control-115.9 2.2 60.1 3.1 9.4 0.8 18.5 3.1 HD1 control-12 5.6 2.1 61.7 2.6 8.60.8 18.6 3.4 HD1 control-13 5.5 2.1 63.2 2.4 8.0 0.8 17.9 3.3 HD1control-14 5.6 2.3 61.7 2.8 7.8 0.8 19.1 2.8 HD1 control-15 5.5 2.4 62.22.4 8.1 0.8 18.5 3.4 HD1 control-16 5.6 2.7 60.8 2.5 8.0 0.9 19.5 3.2HD1 control-avg 5.5 2.3 61.7 2.5 8.0 0.9 19.1 3.2 Fm d15-NY-1 5.9 2.958.3 1.8 12.0 0.9 18.2 6.7 Fm d15-NY-2 5.1 2.2 65.4 1.7 6.1 0.8 18.7 3.5Fm d15-NY-3 5.8 3.0 58.3 2.0 12.4 0.9 17.7 6.3 Fm d15-NY-4 5.1 2.4 62.61.9 8.3 0.9 18.8 4.4 Fm d15-NY-5 5.7 3.0 61.1 1.2 10.7 0.9 17.5 8.9 Fmd15-NY-6 5.4 2.8 58.9 2.5 8.7 0.9 20.7 3.5 Fm d15-NY-7 5.9 2.9 58.7 1.312.2 0.9 18.0 9.2 Fm d15-NY-8 6.2 3.2 57.0 1.5 13.3 0.9 17.8 8.8 Fmd15-NY-9 5.5 2.8 59.7 1.5 10.7 0.9 18.8 7.2 Fm d15-NY-10 5.9 2.6 58.61.4 11.6 0.9 19.0 8.2 Fm d15-NY-11 5.5 2.7 60.0 2.1 9.6 0.9 19.3 4.6 Fmd15-NY-12 5.5 2.6 58.6 2.5 10.7 0.8 19.3 4.2 Fm d15-NY-13 5.5 2.5 59.72.6 9.8 0.8 19.0 3.8 Fm d15-NY-14 5.2 2.8 63.6 2.1 7.4 0.8 18.0 3.5 Fmd15-NY-15 5.9 2.3 61.7 2.4 8.5 0.9 18.3 3.6 Fm d15-NY-16 5.6 3.1 58.02.7 9.3 1.0 20.3 3.5 Fm d15-NY-17 5.7 2.9 60.0 1.4 12.3 0.8 16.9 8.7 Fmd15-NY-18 5.9 3.2 59.2 1.4 11.6 0.8 17.8 8.0 Fm d15-NY-19 5.9 3.2 58.31.6 12.3 0.9 17.7 7.5 Fm d15-NY-20 5.6 2.3 61.8 2.4 8.0 0.9 19.0 3.3 Fmd15-NY-21 6.0 3.0 54.1 3.6 11.4 1.0 20.9 3.1 Fm d15-NY-22 5.9 2.9 61.02.9 7.9 0.8 18.6 2.8 Fm d15-NY-23 6.0 2.7 56.5 1.8 13.0 0.9 19.1 7.4 Fmd15-NY-24 5.3 2.8 61.3 2.1 7.9 0.8 19.7 3.8 Fm d15-NY-25 5.7 3.0 56.43.1 11.5 0.9 19.5 3.7 Fm d15-NY avg 5.7 2.8 59.6 2.1 10.3 0.9 18.8 5.5

Wild type Arabidopsis could also be transformed with the chimericconstructs expressing the Δ15 desaturase in a similar way and seeds fromthose plants will contain higher ALA content than untransformed plants.

Thus, the ratio of of ω3/ω6 fatty acids in plant oil can be improved bytransforming the chimeric Δ15 desaturase gene either into wild typeplants or into plants having reduced 18:2. The latter is the consequenceof the Fusarium Δ15 desaturase being a bifunctional Δ12/Δ15 desaturase.Thus, one skilled in the art can transform the bifunctional Δ12/Δ15desaturase into a mutant plant making little or no LA introduce orco-transform a wild type plant with the bifunctional Δ12/Δ15 desaturaseand a DNA suppression construct designed to suppress the host's nativeΔ12 desaturase gene(s). The native Δ12 desaturase genes include genesencoding both extraplastidic and plastidic Δ12 desaturases.

1. A recombinant construct for altering the total fatty acid profile ofmature seeds of an oilseed plant to produce an oil having an omega 3 toomega 6 ratio greater than 0.4, said construct comprising an isolatednucleic acid fragment selected from the group consisting of: (a) anisolated nucleic acid fragment encoding all or part of the amino acidsequence as set forth in SEQ ID NO:2; (b) an isolated nucleic acidfragment that hybridizes with (a) when washed with 0.1×SSC, 0.1% SDS,65° C.; (c) an isolated nucleic acid fragment encoding an amino acidsequence having at least 46.2% sequence identity with the amino acidsequences set forth in SEQ ID NOs:2, 6, 10, 14, 18 based on the ClustalV method of alignment; or (d) an isolated nucleic acid fragment that iscompletely complementary to (a), (b), or (c) wherein said isolatednucleic acid fragment is operably linked to at least one regulatorysequence.
 2. The recombinant construct of claim 1 wherein the isolatednucleic acid fragment is isolated from Fusarium moniliforme.
 3. Anoilseed plant, plant cell, plant tissue or plant part comprising in itsgenome the recombinant construct of claims 1 or
 2. 4. Seeds obtainedfrom the plant of claim
 3. 5. Oil obtained from the seeds of claim
 4. 6.By-products obtained from the processing of the oil of claim
 5. 7. TheBy-products of claim 6 wherein said By-product is lecithin.
 8. Use ofthe oil of claim 5 in food, animal feed or an industrial application. 9.Use of the By-product of claim 6 or 7 in food or animal feed.
 10. Theoilseed plant of claim 1 or 3 wherein said plant is selected from thegroup consisting of soybean, corn, rapeseed, canola, flax, andsunflower.
 11. A method for increasing the ratio of omega-3 fatty acidsto omega-6 fatty acids in an oilseed plant comprising: a) transformingan oilseed plant cell of with the recombinant construct of claims 1 or2; b) regenerating an oilseed plant from the transformed plant= cell ofstep (a); c) selecting those transformed plants having an increasedratio of omega-3 fatty acids to omega-6 fatty acids compared to theratio of omega-3 fatty acids to omega-6 fatty acids in an untransformedplant.
 12. An oilseed plant made by the method of claim
 11. 13. Seedsobtained from the oilseed plant of claim
 12. 14. Oil obtained from theseeds of claim
 13. 15. By-products obtained from the processing of theoil of claim
 14. 16. The By-products of claim 15 wherein said By-productis lecithin.
 17. Use of the oil of claim 14 in food, animal feed or anindustrial application.
 18. Use of the By-product of claims 15 or 16 infood or animal feed.
 19. The oilseed plant of claim 11 or 12 whereinsaid plant is selected from the group consisting of soybean, corn,rapeseed, canola, flax, and sunflower.
 20. A method for producingalpha-linolenic acid in seed of an oilseed plant wherein thealpha-linolenic acid content of the oil in the seed is at least 25% ofthe total fatty acid content of the seed oil, said method comprising: a)transforming an oilseed plant cell of with the recombinant construct ofclaims 1 or 2; b) regenerating an oilseed plant from the transformedplant cell of step (a); c) selecting those transformed plants having atleast 25% alpha-linolenic acid of the total fatty acid content of theseed oil.
 21. An oilseed plant made by the method of claim
 20. 22. Seedsobtained from the oilseed plant of claim
 21. 23. Oil obtained from theseeds of claim
 22. 24. By-products obtained from the processing of theoil of claim
 23. 25. The By-products of claim 24 wherein said By-productis lecithin.
 26. Use of the oil of claim 23 in food, animal feed or anindustrial application.
 27. Use of the By-product of claims 24 or 25 infood or animal feed.
 28. The oilseed plant of claim 20 or 21 whereinsaid plant is selected from the group consisting of soybean, corn,rapeseed, canola, and sunflower.
 29. A recombinant construct foraltering the total fatty acid profile of mature seeds of an oilseedplant to produce an oil having an omega 3 to omega 6 ratio greater than2, wherein said oil has an eicosapentaenoic acid content greater than2%, said construct comprising an isolated nucleic acid fragment selectedfrom the group consisting of: (a) an isolated nucleic acid fragmentencoding all or part of the amino acid sequence as set forth in SEQ IDNO:2; (b) an isolated nucleic acid fragment that hybridizes with (a)when washed with 0.1×SSC, 0.1% SDS, 65° C.; (c) an isolated nucleic acidfragment encoding an amino acid sequence having at least 46.2% sequenceidentity with the amino acid sequences set forth in SEQ ID NOs:2, 6, 10,14, 18 based on the Clustal V method of alignment; or (d) an isolatednucleic acid fragment that is completely complementary to (a), (b), or(c) wherein said isolated nucleic acid fragment is operably linked to atleast one regulatory sequence.
 30. The recombinant construct of claim 29wherein the isolated nucleic acid fragment is isolated from Fusariummoniliforme.
 31. An oilseed plant, plant cell, plant tissue or plantpart comprising in its genome the recombinant construct of claim
 29. 32.Seeds obtained from the plant of claim
 31. 33. Oil obtained from theseeds of claim
 32. 34. By-products obtained from the processing of theoil of claim
 33. 35. The By-products of claim 34 wherein said By-productis lecithin.
 36. Use of the oil of claim 33 in food, animal feed or anindustrial application.
 37. Use of the By-product of claim 33 or 34 infood or animal feed.
 38. The oilseed plant of claim 29 or 31 whereinsaid plant is selected from the group consisting of soybean, corn,rapeseed, canola, flax, and sunflower.
 39. A method for producingeicosapentaenoic acid in seed of an oilseed plant to produce an oilhaving an omega 3 to omega 6 ratio greater than 2, wherein said oil hasan eicosapentaenoic acid content greater than 2% of the total fatty acidcontent of the seed oil, said method comprising: a) transforming anoilseed plant cell of with the recombinant construct of claim 29; b)regenerating an oilseed plant from the transformed plant cell of step(a); c) selecting those transformed plants having at least 2%eicosapentaenoic acid of the total fatty acid content of the seed oil.40. An oilseed plant made by the method of claim
 39. 41. Seeds obtainedfrom the oilseed plant of claim
 40. 42. Oil obtained from the seeds ofclaim
 41. 43. By-products obtained from the processing of the oil ofclaim
 42. 44. The By-products of claim 43 wherein said By-product islecithin.
 45. Use of the oil of claim 42 in food, animal feed or anindustrial application.
 46. Use of the By-product of claims 44 or 45 infood or animal feed.
 47. The oilseed plant of claim 39 or 40 whereinsaid plant is selected from the group consisting of soybean, corn,rapeseed, canola, flax, and sunflower.